Methods for treating wounds

ABSTRACT

Novel compositions for treating wounds and promoting the healing thereof are described, including composition containing novel combinations of a carrier and recombinant platelet derived grown factor having fewer isoforms and enhanced biostability. Methods of treating wounds with novel therapeutic composition using dosing procedures leading to effective results with a minimal number of treatment applications are also described.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.15/256,339, filed on Sep. 2, 2016, which claims the benefit ofPCT/US2015/055522, with an International Filing Date of Oct. 14, 2014,which claims benefit to U.S. Provisional Application Ser. No.62/063,793, filed Oct. 14, 2014, the disclosures which are all herebyincorporated by reference in their entirety, including all figures,tables, and amino acid or nucleic acid sequences.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 1, 2016, isnamed 50250701301SegList and is 1.27 Kilobytes in size.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to compositions and methods useful fortreating wounds, and in particular, treating hard to heal wounds, suchas lower extremity ulcers in a diabetic patient, venous stasis ulcers,pressure ulcers, severe burns and large surgical wounds such asabdominoplasties and other types of surgical tissue flaps.

BACKGROUND ART OF THE INVENTION

Diabetes is at epidemic proportions worldwide. Reportedly, around 370million people have diabetes and this number is increasing in everycountry. One of the most common and serious complications resulting fromdiabetes is poorly healing wounds that develop most commonly on areas ofhigh pressure on the surface of the foot, such as under the hallux (bigtoe), metatarsophalangeal joints, the tops and ends of the toes, themiddle and sides of the foot and the heel. Foot ulcers form as a resultof nerve damage resulting in a loss of sensation over such pressurepoints on the foot, which leads to extended microtrauma, breakdown ofoverlying tissue, and eventual ulceration. In addition, this loss insensation can allow minor scrapes or cuts to go without proper treatmentand eventually lead to the formation of ulcers. A significant percentageof diabetics will develop a foot ulcer during their lifetime. Once adiabetic foot ulcer (DFU) is formed, treatment can be challenging,particularly in view of the compromised healing environment due to thepresence of neuropathy, vascular disease, altered neutrophil function,diminished tissue perfusion and/or defective protein synthesis, all ofwhich often accompany diabetes.

There is a great need for better treatment modalities for these chroniculcerations. DFUs are a leading cause of amputation. The longer thesewounds remain, the greater the opportunity for them to increase in sizeand depth and become infected. As a consequence, these complicationsresult in 80,000 amputations annually in the U.S. alone. This chronicpathology also severely compromises the overall health of the patientleading to a further downward health spiral of these patients, andadditional costs to the health care system; their treatment doubles thecost of care for affected diabetic patients.

The principle aim of DFU management is wound closure. Under the currentstandard of care, DFU wound care focuses on radical and repeateddebridement, frequent inspection and bacterial control, off-loading ofany pressure on the wound, and careful moisture balance to preventmaceration. Effective DFU healing, however, has not been consistentlyachieved through this approach, and results can depend heavily onpatient compliance. As a result, adjunctive treatments have beendeveloped to address DFUs. Consensus reports for the management ofdiabetic foot ulcers recommend that for ulcers showing less than 50%healing at 4 weeks following good standard wound care, advancedtherapeutics should be considered in order to speed wound healing anddecrease complications. Such advanced treatments include negativepressure wound therapy, biological dressings, bioengineered skinequivalents, hyperbaric oxygen therapy, platelet rich plasma and growthfactors.

However, only a small number of these advanced wound-care products havebeen shown to accelerate DFU healing in prospective, randomizedregistration trials, and even some of those results have been calledinto question by other studies. Among the products that have beenstudied in prospective, randomized registration trials are includedbecaplermin (Regranex®; Smith and Nephew), a topical gel containingrecombinant human platelet-derived growth factor B chain homodimer(rhPDGF-BB), BioChaperone PDGF-BB (Adocia, Lyon, France), a topicalspray that includes molecules that complex with PDGF, two living skinequivalents: a bi-layered skin substitute (Apligraf®; Organogenesis,Inc., Canton, Mass.) and a human fibroblast-derived dermal substitute(Dermagraft®; Shire, Plc., Dublin, Ireland), and vacuum-assisted woundclosure (V.A.C.®; KCl, San Antonio, Tex.). Other treatment modalitiesthat have less rigorous trial data include collagen, platelet-richplasma, silver products, hyperbaric oxygen and electrical stimulation.

Despite some favorable results from prospective, randomized registrationtrials for certain advanced wound-care products, their overall benefitshave been disappointing, as evidenced by the continuing high amputationrates. The following results were reported in a published meta-analysisof 35 randomized, controlled trials evaluating diabetic foot ulceradvanced therapies:

-   -   Platelet-rich plasma did not improve diabetic ulcer healing        compared to good standard wound care.    -   Dermagraft® biological skin equivalent in pooled results from        three studies showed non-significant improvement compared to        standard care, with ulcer healing favoring Dermagraft (35%        versus 24%).    -   Apligraf® biological skin equivalent bi-layer has been reported        to improve healing over good standard wound care (55% versus        34%, p=0.001; 2 studies).    -   5, Regranex® rhPDGF-BB showed improvement in the percentage of        ulcers healed compared to placebo or good standard wound care        (58% versus 37%, p=0.04; 7 studies)    -   V.A.C.® negative pressure wound therapy improved healing more        than good standard wound care (43% versus 29%, p<0.05; 1 study).    -   There was low or insufficient evidence for all studies related        to enhanced time to ulcer healing.        In addition, in four studies the incidence of complete wound        closure for Regranex was reported as 50% or less (48%, 50%, 44%,        and 36%).

Such advanced therapies have not resulted in a consistently effectivesolution to treating DFUs. In view of their mixed clinical results alongwith their greater product cost compared to standard therapy, none ofthese advanced therapies have been widely adopted as a new standard ofcare for treating DFUs.

As noted above, one such advanced therapy is Regranex gel (becaplerman),which consists of rhPDGF-BB at a concentration of 100 μg/g in sodiumcarboxymethylcellulose gel. Specifically, Regranex is formulated as amulti-use, non-sterile, low bioburden, preserved, sodiumcarboxymethylcellulose-based (CMC) topical gel, and is indicated fordaily application to improve the healing of chronic DFUs over severalmonths. The Regranex package insert (label) states that it should beapplied daily up for up to 140 daily applications over a 20 week period,and even longer if the physician deems it appropriate at a dose equal toabout 0.006 mg/cm² (6.25 μg) of wound surface area.

Regranex remains the state-of-the-art growth factor therapy for healingof wounds, as evidenced by the fact that it is the only recombinantgrowth factor product to receive FDA approval for treatment of chronicwounds, even though it was FDA-approved over 15 years ago. Moreover, noone has successfully developed another formulation of Regranex (i.e.rhPDGF-BB) since its FDA approval. While clinical and non-clinical datasupport its clinical use, we believe Regranex has a number oflimitations including: 1) the need for daily applications to the DFU bythe patient, requiring daily wound dressing changes by the patient; 2)the low dosing prescribed in the FDA-approved Instructions for Use,about 0.006 mg (6 μg) per cm² of wound surface area; 3) often imprecisedosing due to the difficulty the patient experiences in visualizing andapplying the gel from a tube (similar to a toothpaste tube) onto thewound which is often located on the bottom of the foot; 4) the need tokeep the product refrigerated (about 2-8° C.); 5) lack of sterility ofthe Regranex gel; 6) the need for prolonged patient use—up to, andpotentially exceeding, 140 daily applications over about a five monthperiod; and 7) the use of the carboxymethylcellulose-based (CMC) topicalgel which lacks the ability to provide a biological matrix for cellularingrowth.

Furthermore, Regranex has been only modestly accepted by the medicalcommunity as an effective treatment for DFUs. Following the EuropeanMedicines Agency (EMA) review of data from four Regranex efficacyclinical trials, the EMA concluded that a 30 μg PDGF/g formulation wasless effective than a 100 μg PDGF/g and there was little differencebetween the 100 μg PDGF/g formulation and a 300 μg PDGF/g formulation.The EMA further concluded that the 100 μg PDGF/g product formulationpossessed only “modest” efficacy.

Perhaps as a result of the “modest” efficacy of Regranex, theeffectiveness of the active ingredient in Regranex (i.e., rhPDGF-BB) intreating wounds has been called into question. Park S A, Raghunathan VK, Shah N M, Teixeira L, Mona M J, et al. (2014) PDGF-BB Does NotAccelerate Healing in Diabetic Mice with Splinted Skin Wounds, PLoS ONE9(8): e104447. doi:10.1371/journal.pone.0104447, reported the resultsfrom a study using a controlled full thickness splinted excisional woundmodel in db/db mice (type 2 diabetic mouse model). Two splinted 8 mmdorsal full thickness wounds were made in db/db mice, and were topicallytreated once daily with either 3 μg PDGF-BB in 30 μl of 5% PEG-PBSvehicle or an equal volume of vehicle for 10 days. The study concludedthat PDGF-BB, although bioactive in vitro, failed to accelerate woundhealing in vivo in the db/db mice using the splinted wound model.

While experts in the field question the effectiveness of Regranex'sactive ingredient, rhPDGF-BB, Applicants believe that there are a numberof reasons for Regranex's questionable efficacy. First, Regranex isdelivered to the wound site by a gel carrier. This formulation allowsthe rhPDGF to be cleared from the site within minutes to hours. Second,while the gel carrier is biocompatible, we believe it provides nosubstrate for cell and vascular ingrowth and in fact may be inhibitoryto cell growth and migration in the wound thereby potentially slowingthe healing process and resulting in suboptimal healing. Third, Regranexis non-sterile, only stable when stored at 2-8 degrees C. (refrigerated)and must be applied daily often to hard to reach anatomical sites, allleading to poor patient compliance; Fourth, although the clinic datashowed no difference between the 100 μg/g formulation and the 300 μg/gformulation, Applicants believe that the growth factor in Regranex is attoo low of a concentration for optimal cell recruitment andproliferation. The Regranex dose per square centimeter of wound surfacearea is only 6 μg and Applicants believe that is too low for optimalcell recruitment and proliferation. Fifth and finally, despite itscommercial use on patients for the past 15 years, the Applicants believethat the growth factor that is included in Regranex is not fully potent.The rhPDGF used in Regranex is recombinantly produced in a yeastexpression system. When expressed in yeast, the protein is excreted as afully folded homodimeric protein consisting of two antiparallel B chainsheld together by two interchain disulfide bonds. However, duringfermentation, internal proteolysis (clipping between residues Arg32 andThr33) and C-terminal truncation (Arg32 and Thr109) may occur. Internalproteolysis yields three potential forms of rhPDGF-BB: intact (both Bchains are intact), single-clipped (one B chain is clipped), anddouble-clipped (both B chains are clipped). Clipping also creates newC-terminal sites for further C-terminal truncations and leads to a verycomplex mixture of isoforms. Applicants believe that the non-intactisoforms of rhPDGF-BB that are included in Regranex are far lesseffective in treating DFUs than the fully intact isoform.

rhPDGF-BB has also been used in orthopedic and periodontal indications,wherein the healing environments and the healing processes are verydifferent from dermal wounds. Two such products include Augment BoneGraft and GEM21S, both of which include rhPDGF-BB as an activeingredient. GEM21S, consisting of rhPDGF-BB solution and a particulatesynthetic bone substitute, was FDA-approved in 2005 and is indicated toimprove bone healing in chronic periodontal defects. Augment Bone Graftalso consists of rhPDGF-BB solution and a particulate synthetic bonesubstitute. Augment is FDA approved, based on a 434 patient pivotalclinical trial in the US and Canada, for improving bone fusion in footand ankle fusion following a single implantation into the bone defectduring surgery. However neither of these products is indicated fortreating dermal wounds, such as DFU's, and both focus on using theproduct to promote bone growth and fusion, a very different cellular andphysiologic process from skin wound healing, through a singleintra-surgical application. Like Regranex, the GEM21S and Augment BoneGraft products must be stored refrigerated (about 2-8° C.) compromisinguser convenience and compliance.

In summary, poor patient outcomes leading to high amputation rates, andconflicting scientific analyses demonstrate that there remains a needfor a more predictable, patient/user friendly and consistently effectivemethod and therapeutic composition for promoting dermal wound healing,including treating DFU's and other types of hard to heal wounds.

DISCLOSURE OF THE INVENTION

The inventive embodiments provided in this Disclosure of the Inventionare meant to be illustrative only and to provide an overview of selectedembodiments disclosed herein. The Disclosure of the Invention, beingillustrative and selective, does not limit the scope of any claim, doesnot provide the entire scope of inventive embodiments disclosed orcontemplated herein, and should not be construed as limiting orconstraining the scope of this disclosure or any claimed inventiveembodiment.

The present invention provides methods and compositions for treating orpromoting the healing of a wound, such as lower extremity ulcers in adiabetic patient, venous stasis ulcers, pressure ulcers, severe burns,traumatic injuries and large surgical wounds such as abdominoplastiesand other types of surgical tissue flaps. In some embodiments, the woundmay extend into the subcutaneous tissue or beyond, or the wound may be adiabetic foot ulcer.

Provided herein is an improved formulation of rhPDGF-BB thatsimultaneously includes a combination of the following improvements andbenefits 1) a carrier that facilitates maintaining an effective PDGFdosage at a wound site for an extended period of time; 2) a carrier thatprovides a substrate for cell attachment and vascular ingrowth; 3) issterile and therefore safer; 4) does not have to be refrigerated and istherefore safer and easier for patients to handle; 5) is applied lessfrequently than current therapies, preferably about once every otherweek, which facilitates better patient compliance and ease of use; 6)has rhPDGF present at a higher concentration than prior artformulations; and 7) contains a more pure and potent rhPDGF-BBformulation with fewer isoforms than certain prior art formulations. Incertain embodiments of the invention, all of the above improvements andbenefits are simultaneously realized.

Provided herein is a method of treating wounds comprising applying atherapeutic composition to the wound surface, monitoring the healing ofthe wound, and periodically reapplying the therapeutic composition tothe wound surface, if deemed necessary, to achieve healing. In someembodiments, the method further includes debriding the wound to removenecrotic or infected tissue before applying the therapeutic compositionand covering the wound with a dressing following the application of thetherapeutic composition. In certain embodiments, a semi-occlusive orocclusive dressing, and the dressing may be periodically changed, suchas changing the dressing with each reapplication of the therapeuticcomposition. The method of the present invention may also include thestep of cleaning the wound at a dressing change with saline or anappropriate antiseptic wound cleansing agent and/or debriding chemicalagent. The methods provided herein may also include treating the patientwith a form of infection control or negative pressure wound therapy.

In some embodiments, the method further comprises forming thetherapeutic composition by combining sterile PDGF and a sterilebiocompatible matrix. The sterile PDGF may be a pre-formulated sterilePDGF solution or it may be formed as part of the treatment procedure byreconstituting a lyophilized sterile powder containing PDGF with asterile water or buffer solution. In some embodiments, the biocompatiblematrix is a sterile porous matrix and may be selected from the groupconsisting of natural polymers such as collagen, gelatin, fibrin,alginate, cellulose, or fibronectin. Alternatively the biocompatiblematrix is a sterile porous matrix selected from the group of syntheticpolymers such as poly(DL-lactide-co-glycolide) (PLGA),poly(DL-lactide)(PDLA), poly(L-lactide)(PLLA),poly(e-caprolactone)(PCL), polyurethane or others. In some embodiments,the biocompatible matrix is a collagen sponge or a mixture of naturaland synthetic polymers.

In some embodiments the therapeutic composition is formed directly onthe wound surface by either first applying a matrix, such as a collagensponge, to the wound surface and then applying a PDGF solution to thecollagen sponge, or alternatively by first applying a PDGF solution tothe wound surface and then applying a matrix, such as a collagen sponge,to the wound surface. In some embodiments the therapeutic composition isformed by first forming a sterile PDGF solution by reconstituting alyophilized sterile powder containing PDGF with a sterile water orbuffer solution, and then aseptically adding the sterile PDGF solutionto a sterile porous biocompatible matrix, such as a collagen sponge, insuch a way that the matrix is wetted with the PDGF solution.

In accordance with another aspect of the present invention, there isprovided herein a method of treating a dermal wound comprising:debriding the wound; applying a therapeutic composition containingrecombinant platelet derived growth factor BB (rhPDGF-BB) to the woundabout once every 3 to 42 days for a treatment period of about 2 to about20 weeks, and wherein said first dose comprises at least about 10 μg ofrhPDGF/cm² of wound surface area; and covering the wound with a dressingfollowing each application of the therapeutic composition. In someembodiment, the method may further comprise advising said patient toavoid applying pressure on the wound as it heals. In some embodimentsthe cumulative total amount of rhPDGF-BB applied to the wound during thetreatment period is less than about 25 mg or about 10 mg or about 5 mgor about 4 mg or about 3 mg or about 2 mg or about 1 mg of rhPDGF-BB. Insome embodiments, the method comprises applying the therapeuticcomposition to the wound once every 7 to 28 days or once every 7 to 21days or once every 10 to 15 days or once every 12 to 14 days. In someembodiments, each treatment includes application of at least about 10 μgof rhPDGF/cm² of wound surface area, or between about 10 μg ofrhPDGF/cm² of wound surface area and about 5,000 μg of rhPDGF/cm² ofwound surface area.

The present invention also provides a therapeutic composition comprisingsterile PDGF and a biocompatible matrix that may be sterile and/orporous. In some embodiments, the sterile PDGF comprises a pre-formulatedsterile PDGF solution, and in other embodiments the sterile PDGFcomprises lyophilized sterile powder containing PDGF reconstituted withsterile water or buffer solution.

In certain embodiments, the sterile PDGF included in the therapeuticcomposition of the present invention comprises an rhPDGF-BB solutioncontaining between about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB. TherhPDGF-BB solution may be formed by combining a sterile powdercontaining lyophilized rhPDGF-BB and sterile water or saline, therebyreconstituting the lyophilized rhPDGF-BB into solution. In certainembodiments, the rhPDGF-BB may be produced through an E. coli expressionsystem wherein at least about 80% of said rhPDGF-BB on a weight basis isunclipped rhPDGF-BB, which may be subsequently lyophilized. In certainembodiments, the lyophilized rhPDGF-BB is capable of being stored atbetween about 20° C. and about 26° C. and still maintain the bioactivityof at least 80% of said rhPDGF-BB for at least about six months or atleast about one year, or between about 16° C. and about 32° C. and stillmaintain the bioactivity of at least 80% of said rhPDGF-BB for at leastabout six months or at least about one year.

In certain embodiments, the matrix included in the therapeuticcomposition of the present invention may be selected from the groupconsisting of collagen, gelatin, fibrin, alginate, cellulose, Chitan orfibronectin. The matrix may provide a resorbable cell scaffold, and maycomprise a collagen sponge. In certain embodiments, the matrix that hasa pore size distribution of between about 10 microns to about 2,000microns, and/or an average pore size of between about 50 microns toabout 500 microns. In certain embodiments, some of the pores areinterconnected or the majority of the pores are interconnected.

Also provided herein is a therapeutic composition comprising anrhPDGF-BB solution and a carrier, such as a matrix, wherein the ratio ofthe rhPDGF-BB solution to the matrix is between about 4 μl/cm³ to about40 ml/cm³ or the ratio of rhPDGF-BB to the matrix is between about 1.2μg PDGF/cm³ of carrier to about 12 mg PDGF/cm³ of carrier. The rhPDGF-BBsolutions disclosed herein may comprise between about 0.05 mg/ml toabout 5 mg/ml or between about 0.1 mg/ml to about 1 mg/ml or betweenabout 0.2 mg/ml to about 0.4 mg/ml of rhPDGF-BB. The rhPDGF-BB solutionsdisclosed herein may comprise about 0.3 mg/ml or about 0.5 mg/ml orabout 1.0 mg/ml of rhPDGF-BB. In certain embodiments, at least about 80%or about 85% or about 90% or about 95% or about 97% of the rhPDGF-BBincluded in the PDGF solution or the therapeutic composition on a weightbasis is unclipped rhPDGF-BB.

In certain aspects, a therapeutic composition is provided comprising arhPDGF-BB solution and a matrix wherein at least about 20% of therhPDGF-BB is entrapped within the matrix′ pores, such that when saidcomposition is applied to a wound on a patient, the rhPDGF-BB isreleased over time as the matrix is absorbed by the patient's body. Incertain embodiments, the therapeutic composition provides sustaineddelivery of rhPDGF-BB at the wound site as the matrix is resorbed andsimultaneously provides a matrix for new cell and tissue ingrowth.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Methods and materials aredescribed herein for use in the present disclosure; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically shows the changes in mean wound area over time inthree groups of animals included in a study as calculated by digitalcaliper measurements.

FIG. 2 graphically shows the changes in mean wound area over time inthree groups of animals included in a study as calculated using ImageJsoftware.

FIG. 3 graphically shows the wound area as calculated using ImageJsoftware for each test animal on day 0 of a study.

FIG. 4 graphically shows the wound area as calculated using ImageJsoftware for each test animal on day 7 of a study.

FIG. 5 graphically shows the wound area as calculated using ImageJsoftware for each test animal on day 14 of a study.

FIG. 6 graphically shows the wound area as calculated using ImageJsoftware for each test animal on day 21 of a study.

FIGS. 7, 7A-7B graphically show that positive results are achieved withfewer applications of the therapeutic composition. FIG. 7A graphicallyshows the amount of wound healing (mm²) using ImageJ software per thenumber cumulative dosages for each of the four time point s (Day 0, Day7, Day 14, and Day 21) for the Regranex Group 1 and rhPDGF/collagenGroup 3. FIG. 7B shows the average percent healing for the RegranexGroup 1, Collagen Group 2, and rhPDGF/collagen Group 3 at each of thefour time points.

FIGS. 8, 8A-D graphically shows the average percent wound closure foreach group over the treatment period: FIG. 8A shows Day 0; FIG. 8B showsDay 7; FIG. 8C shows Day 14; and FIG. 8D shows Day 21.

FIGS. 9-12 include a series of wound images showing the degree ofhealing over time for animals included in a study testing compositionsin accordance with the invention.

FIG. 9 illustrates the state of the wound in 15 test subjects on day 0of the study.

FIG. 10 illustrates the state of the wound in 13 test subjects on day 7of the study.

FIG. 11 illustrates the state of the wound in 13 test subjects on day 14of the study.

FIG. 12 illustrates the state of the wound in 13 test subjects on day 21of the study.

FIGS. 13-18 include a series of photomicrographs of a cross section ofthe wound site on day 21 from three study animals.

FIG. 13 is a photomicrograph (2× magnification) of a study controlanimal treated with a collagen sponge and buffer.

FIG. 14 is a photomicrograph (10× magnification) of a study controlanimal treated with a collagen sponge and buffer.

FIG. 15 is a photomicrograph (2× magnification) of a study animaltreated with a composition in accordance with the present invention.

FIG. 16 is a photomicrograph (10× magnification) of a study animaltreated with a composition in accordance with the present invention.

FIG. 17 is a photomicrograph (2× magnification) of a study animaltreated with Regranex.

FIG. 18 is a photomicrograph (10× magnification) of a study controlanimal treated with Regranex.

MODES FOR CARRYING OUT THE INVENTION

The present invention provides for a novel method of treating dermalwounds, such as diabetic foot ulcers (DFUs), venous stasis ulcers,pressure ulcers, burns, tramatic injuries and large surgical wounds. Thepresent invention additionally provides for novel bioactive therapeuticcompositions for use in treating such wounds, novel methods of preparingbioactive dressings useful for the treatment of wounds, and noveltreatment regimens to improve patient compliance and wound healing.

The novel methods and therapeutic compositions in accordance with thepresent invention will enable equivalent or superior efficacy comparedto prior art products in treating dermal wounds, and provide a bettersafety profile and improved patient compliance and convenience. Thenovel therapeutic compositions provided herein provide: 1) prolongeddelivery of the PDGF onto the wound from each application, thusobviating the need for far more frequent applications by the patient(e.g., daily or every other day applications with prior art products);2) a physical material such as a collagen sponge that can be appliedlike a Band-Aid onto the wound once every several days thus improvingpatient compliance; 3) stability at room temperature, eliminating theneed to keep the product refrigerated; 4) a sterile product improvingsafety over prior art products; 5) a higher initial dose of PDGFcompared to prior art products which better initiates the healingprocess thus reducing the need for prolonged patient use; 6) the use ofan improved carrier that not only sustains the delivery of the PDGF butsimultaneously provides a biological scaffold and/or open porous matrixthat facilitates ingrowth of cells, blood vessels and new tissue leadingto improved healing compared to prior art products which lack theability to provide a biological matrix for cellular ingrowth; and 7)contains a more pure and potent rhPDGF-BB formulation with fewerisoforms than prior art formulations. The novel methods disclosed hereinprovide: 1) a higher initial dose of PDGF as compared to prior artproducts to better initiate the healing process, thus reducing the needfor prolonged patient use; and 2) a treatment protocol that willfacilitate improved patient compliance and convenience by requiringfewer periodic applications of the therapeutic composition, perhaps asfew as 1 to 6 applications versus the 140 applications required by priorart products.

I. Definitions/Nomenclature

As used herein unless otherwise indicated, open terms such as “contain,”“containing,” “include,” “including,” and the like mean comprising.

Some embodiments herein contemplate numerical ranges. When a numericalrange is provided, the range includes the range endpoints unlessotherwise indicated. Unless otherwise indicated, numerical rangesinclude all values and subranges therein as if explicitly written out.

Some values herein are modified by the term “about.” In some instances,the term “about” in relation to a reference numerical value can includea range of values plus or minus 10% from that value. For example theamount “about 10” can include amounts from 9 to 11. In otherembodiments, the term “about” in relation to a reference numerical valuecan include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%,3%, 2%, or 1% from that value.

As used herein, the article “a” means one or more unless explicitlystated otherwise.

Where methods and steps described herein indicate certain eventsoccurring in certain order, those of ordinary skill in the art willrecognize that the ordering of certain steps may be modified and thatsuch modifications are in accordance with the variations of theinvention. Additionally, certain steps may be performed concurrently ina parallel process when possible, as well as performed sequentially.

The meaning of abbreviations is as follows: “C” means Celsius or degreesCelsius, as is clear from its usage, “μL” or “uL” or “ul” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “mm” meansmillimeter(s), “nm” means nanometers, “mM” means millimolar, “μM” or“uM” means micromolar, “M” means molar, “mmol” means millimole(s),“μmol” or “uMol” means micromole(s)”, “g” means gram(s), “μg” or “ug”means microgram(s) and “ng” means nanogram(s), “% w/v” meansweight/volume percent, “% v/v” means volume/volume percent, “HPLC” meanshigh performance liquid chromatography, “UPLC” means ultra performanceliquid chromatography, and “GC” means gas chromatography.

The term “homology” refers to the optimal alignment of sequences (eithernucleotides or amino acids), which may be conducted by computerizedimplementations of algorithms. “Homology”, with regard topolynucleotides, for example, may be determined by analysis with BLASTNversion 2.0 using the default parameters. “Homology”, with respect topolypeptides (i.e., amino acids), may be determined using a program,such as BLASTP version 2.2.2 with the default parameters, which alignsthe polypeptide or fragments (and can also align nucleotide fragments)being compared and determines the extent of amino acid identity orsimilarity between them.

The above descriptions and methods for sequence homology are intended tobe exemplary and it is recognized that this concept is well-understoodin the art. Further, it is appreciated that nucleic acid sequences maybe varied and still provide a functional enzyme, and such variations arewithin the scope of the present invention. The term “enzyme homolog” canalso mean a functional variant.

As used herein, the term “carrier” is intended to refer broadly to anybiologically compatible substance that can serve as a delivery vehiclefor PDGF, whereas the terms “matrix” and “scaffold” are usedinterchangeable to refer to a carrier that acts as a substrate for cellattachment and/or vascular ingrowth as a wound heals, and/or provides ameans for trapping the PDGF within its structure (such as, for example,through interconnected pores), thereby allowing for an ongoing ordelayed or prolonged delivery of PDGF as a wound heals.

II. Novel Method of Treating Wounds

The present invention provides novel methods of treating of wounds. Inone embodiment, a method of treating a wound comprises providing atherapeutic composition comprising a PDGF solution incorporated in abiocompatible scaffold, matrix or carrier and applying the therapeuticcomposition to a wound. A therapeutic composition comprising a PDGFsolution incorporated in a biocompatible scaffold, matrix or carrier,for example, can be applied topically to the wound. In some embodiments,a method of treating a wound comprises multiple periodic applications ofa therapeutic composition to a wound over a period of weeks.

In accordance with one aspect of the present invention, the noveltreatment method for treating wounds includes the following steps:

-   (1) debriding the wound as needed to remove necrotic or infected    tissue;-   (2) forming a therapeutic composition comprising sterile rhPDGF-BB    and a sterile porous biocompatible carrier;-   (3) applying the therapeutic composition containing PDGF to the    wound surface, wherein the carrier provides a substrate for cell    attachment and vascular ingrowth as the wound heals;-   (4) covering the wound with a dressing; and-   (5) monitoring the healing of the wound during a treatment period    and repeating steps (1)-(4) at treatment intervals of 3 or more    days.

The novel treatment method may further include preparing the noveltherapeutic composition prior to applying it to the wound surface,wherein the composition comprises PDGF and a biological matrix. Themethod of preparing the composition may include:

-   (2a) reconstituting a lyophilized (freeze-dried) sterile PDGF powder    with sterile water, saline, a buffer, or a physiologic solution to    provide a specific safe and therapeutic concentration of PDGF; and-   (1b) withdrawing the sterile PDGF solution from a vial (container)    and aseptically adding it to a dry hydrophilic sterile matrix or    patch in such a way that the matrix or patch is wetted with the PDGF    solution.

In some embodiments, the dressing is an occlusive or semi-occlusivedressing. In some embodiments, the repeat of steps (1)-(3) may alsocomprise the steps of: (A) removing the dressing and cleaning the woundwith saline or an appropriate antiseptic wound cleansing agent prior toapplying the therapeutic composition the dressing, and (B) covering thewound with a new dressing following application of the therapeuticcomposition. In some embodiments, novel bioactive therapeuticcompositions described herein may be used in combination with otheraspects of treating wounds, including for example infection control,negative pressure wound therapy, and/or instructing the patient to avoidplacing pressure on the wound site.

In accordance with one aspect of the invention, there is provided atiming schedule for periodically retreating the wound, i.e. repeatingsteps (2)-(4) or periodically reapplying the therapeutic composition tothe wound. The actual number of retreatments and the retreatingfrequency (i.e., the treatment interval) should be determined based on anumber factors including the severity of the wound (e.g., its grade,size and depth), the extent to which the natural wound healingenvironment is compromised (e.g., the vascular supply at the site, themetabolic state of the patient, the ability to off-load pressure on thesite, presence of infection, diabetes stage for a DFU, degree of burnfor a burn), patient's age, duration of the wound, and otherco-morbidities such as smoking, obesity, uncontrolled glucose levels,patient compliance and others. The number of retreatments and theretreatment frequency should be increased for more severe wounds or forwounds with more compromised healing environments. In addition, theprescribed number of treatments and/or the retreatment frequency may beadjusted during the treatment period based on the wound's rate ofhealing, i.e. increase number of retreatments and/or retreatmentfrequency for slower healing wounds, or decrease number of retreatmentsand/or retreatment frequency for faster healing wounds.

In accordance with one aspect of the invention, the retreatmentfrequency is at least about 2 days, at least about 3 days, at leastabout 4 days, at least about 5 days, at least about 6 days, at leastabout 7 days, at least about 8 days, at least about 9 days, at leastabout 10 days, at least about 11 days, at least about 12 days, at leastabout 13 days, at least about 14 days, or at least about 15 days and soon up to at least about once every six weeks, or combinations thereof.In accordance with another aspect of the invention, the retreatmentfrequency is once every 2 to 42 days, or once every 3 to 42 days, oronce every 2 to 28 days, or once every 3 to 28 days, or once every 2 to7 days, or once every 3 to 7 days, or once every 4 to 21 days, onceevery 7 to 28 days, or once every 7 to 21 days, or once every 7 to 14days, or once every 10 to 15 days, or once every 12 to 14 days. Inaccordance with another aspect of the invention, the retreatmentfrequency is once every 2 days, 3 days, 4 days, 5 days, 6 days, 7 days,8 days, 9 days, 10 days, 12 days, 14 days, 15 days, 21 days, 28 days, 30days, 35 days, 42 days, or combinations thereof.

In accordance with one another aspect of the invention, the retreatmentfrequency is substantially the same over the treatment period, and theretreatment frequency is one time at least about every 2 days, at leastabout every 3 days, at least about every 4 days, at least about every 5days, at least about every 6 days, at least about every 7 days, at leastabout every 8 days, at least about 9 every days, at least about every 10days, at least about every 11 days, at least about every 12 days, atleast about every 13 days, at least about every 14 days, or at leastabout every 15 days and so on up to at least about once every six weeks.

In accordance with one aspect of the invention, the wound is retreatedat least 1 time, at least 2 times, at least 3 times, at least 4 times,or at least 5 times over the treatment period. In accordance withanother aspect of the invention, the wound is retreated between 0 and 6times, between 0 and 7 times, or between 0 and 8 times over thetreatment period. In accordance with another aspect of the invention,the wound is treated between 1 to 8 times, or between 2 to 7 times, orbetween 3 to 6 times over the treatment period. In accordance withanother aspect of the invention, the wound is retreated 1, 2, 3, 4, 5,6, 7, 8, 10, or 20 times over the treatment period. In accordance withanother aspect of the invention the wound is retreated between 0 and 46times, or between 1 and 46 times, or between 0 and 20 times, or between1 and 20 times, or between 0 and 27 times, or between 1 and 27 times.

In accordance with one aspect of the invention, the cumulative totalamount of rhPDGF-BB applied to the wound during the treatment period ispreferably more than 0 mg, but less than about 50 mg, or less than about25 mg, or less than about 20 mg, or less than about 15 mg, or less thanabout 10 mg, or less than about 5 mg, or less than about 4 mg, or lessthan about 3 mg, or less than about 2 mg, or less than about 1 mg ofrhPDGF-BB. In certain embodiments, the cumulative total amount ofrhPDGF-BB applied to the wound during the treatment period is preferablybetween about 0.1 mg to about 50 mg, or about 0.5 mg to about 25 mg, orabout 1 mg to about 10 mg, or about 2.5 mg to about 8 mg, or about 3 mgto about 7 mg, about 4 mg to about 6 mg.

The various retreatments may involve the same or different dosages ofrhPDGF-BB, either in terms of the exact amount of rhPDGF-BB that isapplied to the wound (i.e., “absolute dosage”) or in terms of the amountof rhPDGF-BB that is applied per square centimeter (cm²) of wound area(i.e., “area dosage”). In accordance with one aspect of the invention,each treatment applies an absolute dosage of between about 10 μg andabout 50 mg, or between about 10 μg and about 25 mg, or between about 10μg and about 20 mg, or between about 10 μg and about 15 mg, or betweenabout 10 μg and about 10 mg, or between about 10 μg and about 5 mg ofrhPDGF-BB or between about 10 μg and about 1 mg or rhPDGF-BB. Inaccordance with another aspect of the invention, each treatment appliesan area dosage between about 10 μg PDGF/cm² and about 1.0 mg PDGF/cm²,or between about 10 μg PDGF/cm² and about 0.5 mg PDGF/cm², or betweenabout 10 μg PDGF/cm² and about 0.25 mg PDGF/cm², or between about 10 μgPDGF/cm² and 0.1 mg PDGF/cm², or between about 10 μg PDGF/cm² and about0.05 mg PDGF/cm². In certain embodiments, each treatment with rhPDGF-BBis preferably between about 10 μg to 1000 μg PDGF/cm², or about 0.01 mgto about 50 mg PDGF/cm², or about 0.05 mg to about 25 mg PDGF/cm², orabout 0.1 mg to about 10 mg PDGF/cm², or about 0.2 mg to about 2 mgPDGF/cm². In certain embodiments, each treatment applies an area dosagethat is at least about 10 μg of rhPDGF/cm² of wound surface area, or atleast about 25 μg of rhPDGF/cm² of wound surface area, or at least about50 μg of rhPDGF/cm² of wound surface area, or at least about 100 μg ofrhPDGF/cm² of wound surface area, or at least about 250 μg of rhPDGF/cm²of wound surface area, or at least about 500 μg of rhPDGF/cm² of woundsurface area. In certain embodiments, each treatment applies an areadosage that is between about 10 μg of rhPDGF/cm² of wound surface areaand about 500 μg of rhPDGF/cm² of wound surface area, or between about10 μg of rhPDGF/cm² of wound surface area and about 100 μg of rhPDGF/cm²of wound surface area, or between about 15 μg of rhPDGF/cm² of woundsurface area and about 375 μg of rhPDGF/cm² of wound surface area, orbetween about 30 μg of rhPDGF/cm² of wound surface area and about 190 μgof rhPDGF/cm² of wound surface area, or between about 30 μg ofrhPDGF/cm² of wound surface area and about 300 μg of rhPDGF/cm² of woundsurface area.

In accordance with one aspect of the invention, the initial treatmentwith compositions in accordance with the present invention may be themost important treatment. PDGF facilitates the wound healing processthrough its effect on cell proliferation (mitogenesis) and directedcellular movement (chemotaxis) as well as re-vascularization (generatingnew blood vessels). Many cells have been shown to possess receptors(binding sites) for PDGF including connective tissue cells (skin, bone,cartilage, tendon and ligament), blood vessel cells and cells of thenervous system. Cells that possess receptors for PDGF respond bymigrating toward the site of the wound (where PDGF is present atelevated levels as a result of applying therapeutic compositions inaccordance with the present invention) and subsequently proliferatingafter binding PDFG. Since the PDGF receptor is degraded quickly afteractivation, cell proliferation is controlled and limited by the presenceof locally available PDGF as well as by cell-cell interaction that leadscells to proceed from the proliferative phase of wound healing to thatof matrix deposition that ultimately results in complete healing. As aresult, a critical bolus of rhPDGF-BB must be applied during the initialtreatment to ensure that the patient's natural wound healing process isproperly activated. Therefore, in accordance with the invention theinitial treatment comprises applying a therapeutic compositioncontaining an area dosage that is at least 10 μg PDGF/cm² wound surfacearea, up to 5000 μg PDGF/cm² wound surface area, or at least 20 μgPDGF/cm² up to 1000 μg PDGF/cm² wound surface area, or at least 30 μgPDGF/cm² up to 600 μg PDGF/cm² wound surface area, or at least 40 μgPDGF/cm² up to 400 μg PDGF/cm² wound surface area, or at least 50 μgPDGF/cm² up to 350 μg PDGF/cm² wound surface area, or at least 60 μgPDGF/cm² up to 300 μg PDGF/cm² wound surface area, or at least 200 μgPDGF/cm² up to 2000 μg PDGF/cm² wound surface area. In accordance withanother aspect of the invention the initial treatment comprises applyinga therapeutic composition containing an area dosage that is at least 10μg PDGF/cm² wound surface area, or at least 20 μg PDGF/cm² wound surfacearea, or at least 25 μg PDGF/cm² wound surface area, or at least 30 μgPDGF/cm² wound surface area, or at least 40 μg PDGF/cm² wound surfacearea, or at least 50 μg PDGF/cm² wound surface area, or at least 60 μgPDGF/cm² wound surface area, or at least 70 μg PDGF/cm² wound surfacearea, or at least 80 μg PDGF/cm² wound surface area, or at least 90 μgPDGF/cm² wound surface area, or at least 100 μg PDGF/cm² wound surfacearea, or at least 250 μg PDGF/cm² wound surface area, or at least 500 μgPDGF/cm² wound surface area.

In accordance with another aspect of the invention, each treatmentapplies is between about 4 μl PDGF solution/cm³ of carrier (which may bea matrix such as a collagen sponge) to about 40 ml PDGF solution/cm³ ofcarrier, or between about 0.1 ml PDGF solution/cm³ of carrier to about30 ml PDGF solution/cm³ of carrier, or between about 0.2 ml PDGFsolution/cm³ of carrier to about 20 ml PDGF solution/cm³ of carrier, orbetween about 0.1 ml PDGF solution/cm³ of carrier to about 10 ml PDGFsolution/cm³ of carrier, or between about 0.25 ml PDGF solution/cm³ ofcarrier to about 5 ml PDGF solution/cm³ of carrier, or between about0.25 ml PDGF solution/cm³ of carrier to about 2.5 ml PDGF solution/cm³of carrier, or between about 0.1 ml PDGF solution/cm³ of carrier toabout 1 ml PDGF solution/cm³ of carrier, or between about 0.5 ml PDGFsolution/cm³ of carrier to about 1.5 ml PDGF solution/cm³ of carrier. Incertain embodiments, the PDGF solution contains about 0.3 mg/ml ofrhPDGF-BB.

In accordance with another aspect of the invention, each treatmentapplies between about 1.2 μg PDGF/cm³ of carrier to about 12 mg PDGF/cm³of carrier, or between about 30 μg PDGF/cm³ of carrier to about 9 mgPDGF/cm³ of carrier, or between about 60 μg PDGF/cm³ of carrier to about6 mg PDGF/cm³ of carrier, or between about 75 μg PDGF/cm³ of carrier toabout 3 mg PDGF/cm³ of carrier, or between about 75 μg PDGF/cm³ ofcarrier to about 1.5 mg PDGF/cm³ of carrier, or between about 75 μgPDGF/cm³ of carrier to about 750 μg PDGF/cm³ of carrier, or betweenabout 120 μg PDGF/cm³ of carrier to about 600 μg PDGF/cm³ of carrier, orbetween about 150 μg PDGF/cm³ of carrier to about 450 μg PDGF/cm³ ofcarrier, or between about 75 μg PDGF/cm³ of carrier to about 225 μgPDGF/cm³ of carrier.

In accordance with one aspect of the invention, the initial PDGFtreatment absolute dosage may be greater than the subsequent retreatmentdosages. The initial PDGF treatment absolute dosage may be about 10%,about 20%, about 30%, about 40%, or about 50% higher, or up to about300% higher than each of the subsequent retreatment PDGF dosages.

In accordance with one aspect of the invention, the method includesstoring the PDGF at room temperature, generally between 16 and 32degrees C. Prior to use it may be reconstituted with sterile water,saline, a buffer, or other physiologic solution to form a solutionhaving the desired PDGF concentration. The solution is then added to acarrier, preferably a cell matrix (e.g., a collagen sponge) having thedesired porosity in the desired volume to wet the matrix. The rhPDGFsoaked matrix is then applied to the wound surface. If the wound is anexternal wound it is then covered with a wound dressing. This processmay then be repeated in accordance with frequency and durationparameters described above until the wound is substantially healed.

III. Novel Therapeutic Compositions for Treating Wounds

The present invention also provides novel therapeutic compositions fortreating wounds, which comprise sterile PDGF incorporated in abiocompatible sterile carrier, matrix or scaffold. For example, thetherapeutic composition can be applied topically to a wound tofacilitate the wound's healing.

In accordance with one aspect of the invention, a therapeuticcomposition is provided that comprises a rhPDGF-BB solution and acarrier that is preferably a biocompatible cell scaffold, wherein therhPDGF-BB solution is disposed in or incorporated into the cellscaffold. In some embodiments, the rhPDGF-BB solution comprises betweenabout 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, or between about 0.1mg/ml to about 1 mg/ml of rhPDGF-BB, or between about 0.2 mg/ml to about0.4 mg/ml of rhPDGF-BB. In accordance with one aspect of the invention,the rhPDGF-BB solution contains rhPDGF-BB at a concentration of about0.05 mg/ml, or about 0.1 mg/ml, or about 0.2 mg/ml, or about 0.25 mg/ml,or about 0.3 mg/ml, or about 0.35 mg/ml, or about 0.4 mg/ml, or about0.5 mg/ml, or about 0.6 mg/ml, or about 0.7 mg/ml, or about 0.8 mg/ml,or about 0.9 mg/ml, or about 1 mg/ml, or about 2 mg/ml, or about 3mg/ml, or about 4 mg/ml, or about 5 mg/ml.

In some embodiments, the rhPDGF-BB solution is a preformulated asepticPDGF solution comprising the elements described herein (e.g., PDGFconcentration, sterile solution composition, etc.). In other embodimentsthe rhPDGF-BB solution is formed at the time of use, preferably bycombining a sterile solution (e.g., sterile water, saline, a buffersolution, or a physiologic solution) with a sterile powder comprising orconsisting essentially of lyophilized rhPDGF-BB. The sterile solution isused to reconstitute the lyophilized rhPDGF-BB. The lyophilizedrhPDGF-BB is formed by lyophilizing liquid rhPDGF-BB produced by using arecombinant expression system as described further herein below underaseptic conditions.

In another aspect of the invention rhPDGF may be incorporated intocarrier, preferably a sterile, biocompatible, absorbable cell scaffold,and the PDGF saturated carrier is then lyophilized to form a sterile,dry device incorporating rhPDGF. Any known technique for lyophilizingrecombinant proteins may be used to lyophilize rhPDGF-BB so long as ityields a sterile powder. The resulting lyophilized rhPDGF-BB powder iscapable of being stored at room temperature and still maintain at leastabout 80% of its bioactivity for at least about 6 months, or at leastabout 1 year, or at least about 2 years, or at least about 3 years. Thesterile lyophilized device may then be applied directly to a wound siteor wetted either by blood or other sterile solution prior to placementon the wound.

Because PDGF has a tendency to adhere to surfaces of a container, suchas a vial, (particularly at higher pH's) achieving reconstitution of100% of the lyophilized PDGF in a vial may be challenging. Therefore, incertain embodiments, additives may be included in the PDGF solution tolower its pH below about 7, or below about 6, or below about 5 or belowabout 4 or below about 3. Additives that may facilitate reconstitutingthe lyophilized PDGF include salts, carrier proteins such as albumin, orlow pH solutions such as dilute acetic acid or hydrochloric acid. If thePDGF solution is too acidic, however, it could negatively impact thebiocompatible scaffold. Therefore, in certain embodiments, thelyophilized PDGF is reconstituted in a solution having a pH below about5, and once the PDGF is substantially fully reconstituted a basesolution is added to increase the pH of the PDGF solution to betweenabout 6 to about 8, or to increase it to about 7 before it is combinedwith the biocompatible scaffold. Such a pH adjustment step isparticularly useful when the biocompatible scaffold is a collagensponge.

The buffer solution used to reconstitute the lyophilized rhPDGF-BB maycomprise, but is not limited to, water, saline, carbonates, phosphates(e.g. phosphate buffered saline), histidine, acetates (e.g. sodiumacetate), acidic buffers such as acetic acid and HCl, and organicbuffers such as lysine, Tris buffers (e.g.tris(hydroxymethyl)aminoethane),N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), and3-(N-morpholino) propanesulfonic acid (MOPS). Preferably, the buffersolution is sterile. Buffers can be selected based on biocompatibilitywith PDGF and the buffer's ability to impede undesirable proteinmodification. Buffers can additionally be selected based oncompatibility with wound tissues. In one embodiment, sodium acetatebuffer is used. The buffers can be employed at different molarities, forexample, about 0.1 mM to about 100 mM, about 1 mM to about 50 mM, about5 mM to about 40 mM, about 10 mM to about 30 mM, or about 15 mM to about25 mM, or any molarity within these ranges. In some embodiments, anacetate buffer is employed at a molarity of about 20 mM.

As noted above, the rhPDGF-BB solution is combined with carrier to forma therapeutic composition. The carrier may be a matrix or scaffold thatacts as a substrate for cell attachment and/or vascular ingrowth as awound heals, and/or provides a means for trapping the PDGF within itsstructure (such as, for example, through interconnected pores), therebyallowing for an ongoing or delayed or prolonged delivery of PDGF as awound heals and the matrix or scaffold is resorbed by the body. In someembodiments, the carrier or matrix is a biocompatible, resorbable cellscaffold. The carrier or matrix may comprise natural polymers such ascollagen, gelatin, fibrin, alginate, cellulose, Chitosan or fibronectin.The carrier or matrix may also comprise synthetic biocompatible polymersselected from the group of synthetic polymers such aspoly(DL-lactide-co-glycolide) (PLGA), poly(DL-lactide)(PDLA),poly(L-lactide)(PLLA), poly(e-caprolactone)(PCL), polyurethane orothers. The carrier or matrix may also be a mixture of such natural andsynthetic polymers. In some embodiments, the matrix comprises a collagenor gelatin sponge, which may be a Type 1 collagen sponge. A collagensponge holds the rhPDGF at the wound site and concurrently provides ascaffold for cell growth, resulting in improved user friendliness andmore rapid and complete healing. In one aspect the invention, thecarrier or matrix, which may be a collagen sponge, has a porosity ofbetween about 10 microns to about 2 mm, or about 50 microns to about1000 microns, or about 100 microns to about 500 microns. The averagepore size may be between about 50 microns to about 500 microns andwherein the majority of the pores are interconnected.

In some embodiments, carrier or matrix materials are bioresorbable. Acarrier or matrix material, in one embodiment, can be at least 20%, 30%,40%, 50%, 60%, 70%, 75%, 90% or 100% resorbed within one monthsubsequent to its application to the wound. Bioresorbability will bedependent on: (1) the nature of the material (i.e., its chemical makeup,physical structure and size); (2) the location within the body in whichthe material is placed; (3) the amount of material that is used; (4) themetabolic state of the patient (diabetic/non-diabetic, smoker, old age,etc.); and (5) the extent and/or type of wound treated.

In one aspect of the invention, the rhPDGF-BB solution and the carriershould be combined in an appropriate ratio in order to form atherapeutic composition that has optimal effectiveness in healingwounds. In some embodiments, the rhPDGF-BB solution and the carrier arecombined at a ratio that is between about 4 μl PDGF solution/cm³ ofcarrier (which may be a matrix such as a collagen sponge) to about 40 mlPDGF solution/cm³ of carrier, or between about 0.1 ml PDGF solution/cm³of carrier to about 30 ml PDGF solution/cm³ of carrier, or between about0.2 ml PDGF solution/cm³ of carrier to about 20 ml PDGF solution/cm³ ofcarrier, or between about 0.1 ml PDGF solution/cm³ of carrier to about10 ml PDGF solution/cm³ of carrier, or between about 0.25 ml PDGFsolution/cm³ of carrier to about 5 ml PDGF solution/cm³ of carrier, orbetween about 0.25 ml PDGF solution/cm³ of carrier to about 2.5 ml PDGFsolution/cm³ of carrier, or between about 0.1 ml PDGF solution/cm³ ofcarrier to about 1 ml PDGF solution/cm³ of carrier, or between about 0.5ml PDGF solution/cm³ of carrier to about 1.5 ml PDGF solution/cm³ ofcarrier.

In some embodiments, the rhPDGF-BB and the carrier are combined at aratio that is between about 1.2 μg PDGF/cm³ of carrier to about 12 mgPDGF/cm³ of carrier, or between about 30 μg PDGF/cm³ of carrier to about9 mg PDGF/cm³ of carrier, or between about 60 μg PDGF/cm³ of carrier toabout 6 mg PDGF/cm³ of carrier, or between about 75 μg PDGF/cm³ ofcarrier to about 3 mg PDGF/cm³ of carrier, or between about 75 μgPDGF/cm³ of carrier to about 1.5 mg PDGF/cm³ of carrier, or betweenabout 75 μg PDGF/cm³ of carrier to about 750 μg PDGF/cm³ of carrier, orbetween about 120 μg PDGF/cm³ of carrier to about 600 μg PDGF/cm³ ofcarrier, or between about 150 μg PDGF/cm³ of carrier to about 450 μgPDGF/cm³ of carrier, or between about 75 μg PDGF/cm³ of carrier to about225 μg PDGF/cm³ of carrier.

In one aspect of the invention, the carrier is a scaffold and therhPDGF-BB/scaffold ratio is such that when the rhPDGF-BB solution andthe scaffold are combined, the scaffold is capable of entrapping atleast about 20%, 30%, 40% or 50% up to at least about 100% of therhPDGF-BB within the scaffold's pores such that the rhPDGF-BB isreleased over time as the scaffold is absorbed by the patient's body,thereby providing controlled delivery of rhPDGF-BB at the wound siteover an extended period of time and simultaneously providing a matrixfor new cell and tissue ingrowth. In some embodiments, the scaffold iscapable of entrapping between about 20% to about 100%, or between about25% to about 95%, or between 30% to about 90% of the rhPDGF-BB withinthe scaffold's pores. The percentages of PDGF entrapment described aboveare also applicable to entrapment of reconstituted lyophilized PDGF-BB.

Various amounts of rhPDGF-BB may be used in the therapeutic compositionsof the present invention. In accordance with one aspect of theinvention, the total amount of rhPDGF-BB included in the therapeuticcomposition is less than 50 mg, or less than 25 mg, or less or less than10 mg, or less than 5 mg, or less than 2.5 mg or less than 1 mg. Inaccordance with another aspect of the invention the total amount ofrhPDGF-BB included in the therapeutic composition is about 50 mg, orabout 25 mg, or about 10 mg, or about 1.0 mg, or about 0.5 mg, or about0.1 mg.

The concentration of PDGF in embodiments of the present invention can bedetermined by using an enzyme-linked immunoassay as described in U.S.Pat. Nos. 6,221,625, 5,747,273, and 5,290,708, incorporated herein byreference, or any other assay known in the art for determining PDGFconcentration. The concentration of PDGF in the embodiments of thepresent invention is less than about 10 mg/g, or less than about 5 mg/gor less than about 1 mg/g or less than about 0.5 mg/g or less than about0.1 mg/g or less than about 0.05 mg/ml. In another aspect of theinvention the concentration of PDGF in the embodiments of the presentinvention is between about 0.05 mg/g to about 5 mg/g, or between about0.1 mg/g to about 1 mg/g or between about 0.25 mg/g and about 0.5 mg/g.

The PDGF-BB used in the therapeutic composition of the present inventionmay be derived from any source such as natural source, synthetic sourceor recombinant source. In accordance with one aspect of the invention,PDGF is produced by recombinant DNA techniques. When PDGF is produced byrecombinant DNA techniques, a DNA sequence encoding a single monomer(e.g., PDGF B-chain), is inserted into cultured cells for expression ofthe B chain monomer. The monomer is then extracted and isolated from thecell culture and refolded to form the biologically active homodimer(e.g., PDGF-BB), which may be further processed for additionalpurification. In accordance with one aspect of the invention, thecultured cells are prokaryotic cells or are E. coli cells. The rhPDGF-BBproduced through these recombinant techniques can be purified inaccordance with the techniques outlined in PCT No. WO 2005/077973, whichis incorporated herein.

As noted above, prior art recombinant DNA production methods haveresulted in mixtures of rhPDGF-BB fragments. In accordance with oneaspect of the invention, substantially all of the rhPDGF-BB included inthe therapeutic compositions described herein are intact non-clippedchains. In accordance with one aspect of the invention, the bacterialexpression system is an E. coli expression system, and the resultingprotein is purified using reversed phase high performance liquidchromatography, gel filtration or ion exchange chromatography, or somecombination thereof, wherein the resulting rhPDGF-BB contained in thepurified protein composition is at least about 80%, or at least about85%, or at least about 90%, or at least about 95%, or at least about 97%unclipped rhPDGF-BB on a weight basis.

In some embodiments, the rhPDGF-BB included in the therapeuticcompositions of the present invention is a rhPDGF-BB that comprises orconsists essentially of an amino acid sequence having at least about90%, about 92%, about 94%, about 96%, about 98%, about 99%, or about100% homology to SEQ ID NO. 1, which is provided below:

SEQ ID NO. 1: Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala Met Ile                  5                  10        Ala Glu Cys Lys Thr Arg Thr Glu Val Phe Glu Ile Ser     15                  20                  25Arg Arg Leu Ile Asp Arg Thr Asn Ala Asn Phe Leu Val             30                  35Trp Pro Pro Cys Val Glu Val Gln Arg Cys Ser Gly Cys 40                  45                  50Cys Asn Asn Arg Asn Val Gln Cys Arg Pro Thr Gln Val         55                  60                  65Gln Leu Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val                 70                  75Arg Lys Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu     80                  85                  90Glu Asp His Leu Ala Cys Lys Cys Glu Thr Val Ala Ala             95                 100 Ala Arg Pro Val Thr 105

In accordance with another aspect of the invention, the rhPDGF-BBincluded in the therapeutic compositions of the present inventioncomprises or consists essentially of at least about 80%, or at leastabout 85%, or at least about 90%, or at least about 95%, or at leastabout 97% unclipped rhPDGF-BB on a weight basis. In accordance withanother aspect of the invention, the rhPDGF-BB included in thetherapeutic compositions of the present invention comprises or consistsessentially of at least about 80%, or at least about 85%, or at leastabout 90%, or at least about 95%, or at least about 97% of rhPDGF-BBthat comprises or consists essentially of an amino acid sequence havingat least about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologyto SEQ ID NO. 1.

In some embodiments, the components comprising the novel compositions ofthe present invention are provided in a kit. A kit can comprise threecomponents:

a) vial of sterile rhPDGF-BB lyophilized powder,

b) vial of sterile water, a buffer, saline, or a physiologic solution,and

c) a carrier.

The kit will may be stored at room temperature for up to 3 years. Insome embodiments the storage is between 16 and 32 degrees C. In someembodiments, the powder included in the kit comprises a predeterminedamount of PDGF. In some embodiments, the amount of PDGF is consistentwith the values provided herein. In some embodiments, the carrier isincluded in a blister pack comprising a predetermined amount of carrier.In some embodiments, the amount of carrier is consistent with the valuesprovided herein, and the type carrier is consistent with the materialsdescribed herein.

At the time of use, the rhPDGF-BB in the kit will be reconstituted withthe sterile water, saline, buffer, or physiologic solution and thecarrier will be shaped to the size of the wound. Following trimming thecarrier to fit the wound, it will be soaked with the rhPDGF solutionsuch that the solution fully saturates the interior pores of thecarrier. The rhPDGF-saturated carrier will then be applied to thedebrided wound and covered with a wound dressing. This process isrepeated in accordance with the timing schedules described hereinabove.

IV. Methods of Treating Various Types of Wounds

The methods and compositions of the present invention are useful intreating a variety of wounds including diabetic ulcers, pressure ulcers,neuropathic ulcers, vascular ulcers, burns, accidental acute wounds andsurgical wounds. Various wound classification systems exists and can beused to identify wounds that methods and compositions of the presentinvention are particularly useful in treating. Two such ulcerclassification systems include the Wagner classification system (see,Wagner (1987) Orthopedics 10:163-72) and the University of Texasclassification system (see, Lavery (1996) J Foot Ankle Surg 35:528-31).The Wagner system grades the wound by the depth of the wound and thepresence of infection. It has five numeric grades:

Grade 1: Superficial Diabetic Ulcer

Grade 2: Ulcer extension

-   -   Involves ligament, tendon, joint capsule or fascia    -   No abscess or Osteomyelitis

Grade 3: Deep ulcer with abscess or Osteomyelitis

Grade 4: Gangrene to portion of forefoot

Grade 5: Extensive gangrene of foot

The University of Texas classification has four numeric grades based onthe depth of the wound. In addition there are four letter grades, A toD, related to infection and ischemia. The University of Texasclassification system includes:

Stages

-   -   Stage A: No infection or ischemia    -   Stage B: Infection present    -   Stage C: Ischemia present    -   Stage D: Infection and ischemia present

Grading

-   -   Grade 0: Epithelialized wound    -   Grade 1: Superficial wound    -   Grade 2: Wound penetrates to tendon or capsule    -   Grade 3: Wound penetrates to bone or joint        A wound with a numeric grade of 3 and letter grade of D, for        example, would be a wound that penetrates to bone or joint and        is infected and ischemic. In accordance with one aspect of the        present invention, the methods and compositions of the present        invention are used to treat a wound that is either a grade 2,        grade 3 or grade 4 wound under the Wagner classification system,        or a grade 1, 2 or 3 wound (stages A, B, C, or D) under the        University of Texas classification system.

In some embodiments, the methods and compositions described herein maybe used to treat wounds such as lower extremity ulcers, and inparticular foot ulcers on diabetic patients. The methods andcompositions of the present invention are particularly useful intreating non-healing lower extremity diabetic ulcers which have failedto heal by about 50% after about 4 weeks of conventional therapies underthe current standard of care as described above in the Background.

In some embodiments, the compositions of the present invention are usedto treat burns in combination with a 1:1.5 or 1:1.3 meshed splitthickness skin graft (the meshing allows the graft to cover a wider areabut leaves small openings that need to heal), abdominoplasties(so-called “tummy tucks”), healing following other types of plastic andreconstructive surgeries, or post-amputation wounds.

V. Additional Therapeutic Elements

The therapeutic compositions of the present invention may includeadditional therapeutics elements to further facilitate healing a wound.In some embodiments, solutions comprising PDGF can further compriseadditional components, such as other biologically active agents. Inother embodiments, solutions comprising PDGF can further comprise cellculture media, other stabilizing proteins such as albumin, antibacterialagents, protease inhibitors [e.g., ethylenediaminetetraacetic acid(EDTA), ethylene glycol-bis(beta-aminoethylether)-N,N,N′,N′-tetraaceticacid (EGTA), aprotinin, ε-aminocaproic acid (EACA), etc.] and/or othergrowth factors such as fibroblast growth factors (FGFs), epidermalgrowth factors (EGFs), transforming growth factors (TGFs), keratinocytegrowth factors (KGFs), insulin-like growth factors (IGFs), or otherPDGFs including compositions of PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CCand/or PDGF-DD. In addition, biologically active agents that can beincorporated into compositions of the present invention in addition toPDGF can comprise organic molecules, inorganic materials, proteins,peptides, nucleic acids (e.g., genes, gene fragments, small insertribonucleic acids [si-RNAs], gene regulatory sequences, nucleartranscriptional factors, and antisense molecules), nucleoproteins,polysaccharides (e.g., heparin), glycoproteins, and lipoproteins.Additional non-limiting examples of biologically active compounds thatcan be incorporated into compositions of the present invention,including, e.g., anti-cancer agents, antibiotics, analgesics,anti-inflammatory agents, immunosuppressants, enzyme inhibitors,antihistamines, hormones, muscle relaxants, prostaglandins, trophicfactors, growth factors, and vaccines, are disclosed in U.S. patentapplication Ser. No. 11/159,533 (Publication No: 20060084602).

Standard protocols and regimens for delivery of additional biologicallyactive agents are known in the art. Additional biologically activeagents can be introduced into compositions of the present invention inamounts that allow delivery of an appropriate dosage of the agent to thewound site. In most cases, dosages are determined using guidelines knownto practitioners and applicable to the particular agent in question. Theamount of an additional biologically active agent to be included in acomposition of the present invention can depend on such variables as thetype and extent of the condition, the overall health status of theparticular patient, the formulation of the biologically active agent,release kinetics, and the bioresorbability of the biocompatiblescaffold. Standard clinical trials may be used to optimize the dose anddosing frequency for any particular additional biologically activeagent.

EXAMPLES Example 1

The efficacy of a collagen wound dressing containing 0.3 mg/mlrecombinant human platelet derived growth factor-BB (rhPDGF-BB) wasevaluated in the treatment of surgically induced full thickness woundsin mice rendered diabetic by a mutation in the leptin receptor (db/db).

A. Study Design

Fifteen (15) male C57/B6 (Leprdb) db/db mice with an average startingbody weight of 41.46 g were obtained from Jackson Laboratory (BarHarbor, Me.) strain code 000642. Animals were acclimatized prior tostudy commencement. During this period of 3 days, the animals wereobserved daily in order to reject animals that presented in poorcondition.

During the study all animals were single housed under identicalconditions in disposable cages. The study was performed in animal roomsprovided with HEPA-filtered air at a temperature of 70° F.+/−5° F. andrelative humidity of 50%+/−20%. Animal rooms were set to maintain aminimum of 12 to 15 air changes per hour. The room was on an automatictimer for a light/dark cycle of 12 hours on and 12 hours off with notwilight. AlphaDry® bedding was used. Bedding was changed a minimum ofonce per week. Cages, tops, bottles, etc. were washed with a commercialdetergent and allowed to air dry. A commercial disinfectant was used todisinfect surfaces and materials introduced into the hood. Floors wereswept daily and mopped a minimum of twice weekly with a commercialdetergent. Walls and cage racks were sponged a minimum of once per monthwith a dilute bleach solution. A cage card or label with the appropriateinformation necessary to identify the study, dose, animal number andtreatment group marked all cages. The temperature and relative humiditywas recorded during the study, and the records retained. Animals werefed with a sterile Purina Labdiet® 5053 rodent diet and sterilized waterwas provided ad libitum.

At the commencement of the study, the fifteen (15) animals were randomlyand prospectively divided into three (3) groups of five (5) animalseach:

-   -   Group 1—Regranex Gel 0.01% rhPDGF-BB was applied daily for 21        days as prescribed by the package insert;    -   Group 2—a collagen wound dressing combined with buffer was        applied on days 0, 7 and 14; and    -   Group 3—a collagen wound dressing containing 0.3 mg/ml        recombinant human platelet derived growth factor-BB (rhPDGF-BB)        was applied on days 0, 7 and 14.        Each animal was identified by an ear punch corresponding to an        individual number. On Day 0, mean starting weights were        recorded, to ensure that mean starting weights were comparable        among groups. A cage card was used to identify each cage or        label marked with the study number (LYN-01), treatment group        number, and animal numbers.

Test and control collagen+/−PDGF articles were administered topically assurgical dressings (as described below) immediately following theinduction of the wound and were changed every seven (Q7) days. Regranextreated sites were treated as prescribed in the Instructions For Use(IFU) included in the product insert including daily dosing as outlinedbelow. All dressings were applied and held in place using Tegaderm™ andsecured in place outside of the wound area with benzoin. At the time ofdressing change, the wound area was rinsed with saline and the rinse wascollected and stored at −80° C. for future analysis of proteaseactivity. For sites treated with the collagen wound dressing, allnon-adherent collagen was gently removed from the healing wound, thesite rinsed with saline and the rinse collected as described. Followingremoval of the dressing and collection of the rinse, the wound wasmeasured using a caliper and photographed prior to re-application ofdressing/test article. All wound areas were reported in mm².

For photographic documentation of wound healing, the camera was mountedon a tripod at an optimal distance to ensure all photos were consistent.A ruler was placed such that it was captured in the image to allowaccurate estimation of lesion size. In addition to in life measurementsof the wound area, all photographs of the wounds were analyzed usingImage J Software and the wound area was traced and quantitated at theconclusion of the study.

Blood glucose levels were determined prior to the start of the study andagain just prior to sacrifice on Day 21 to confirm diabetic diseasestate. At study termination, the wound site was collected in 10% NBF andprepared for histopathology. The study design is summarized below inTable 1.

TABLE 1 Study Design Group Number of Wound Route/ Wound Number Animals(Day 0) Treatment* Frequency Assessment 1 5 male 1.5 cm × Regranex GelTopical- 21 daily Daily To (db/db) 1.5 cm applications; dose Day 21applied based on open wound measurements at Day 0, 7 and 14** 2 5 male1.5 cm × Collagen wound Topical- 3 weekly Every 7 Days (db/db) 1.5 cmdressing + Buffer applications; Days To Day 21 0, 7 and 14*** 3 5 male1.5 cm × PDGF Bioactive Topical- 3 weekly Every 7 Days (db/db) 1.5 cmWound Dressing applications; Days To Day 21 0, 7 and 14*** *Alldressings were applied and held in place by Tegaderm ™ **The cm lengthof Regranex applied was based on open wound measurements obtained onDays 0, 7 and 14. The centimeter length of Regranex applied daily wasthe same for Days 0-6 and was based on measurements obtained on Day 0.The centimeter length of Regranex applied daily for Days 7-13 was basedon measurements obtained on Day 7 and the centimeter length of Regranexapplied on Days 14-21 was based on measurements obtained on Day 14. See“PDGF-BB Calculations” for detail. ***The volume of PDGF-BB orbuffer/sterile saline that was applied to the collagen sponge at Days 0,7 and 14 utilized the formula: 145 × cm² open wound surface area (length[cm] × width [cm] open wound)

B. Test Articles & Vehicle Preparation

The topical formulations used in the study were Regranex Gel (0.01%rhPDGF-BB in carboxymethylcellulose gel) (Group 1); a collagen wounddressing wetted with rhPDGF-BB (Group 3); and a collagen wound dressingwetted with saline (Group 2). All dressings were covered with Tegaderm™and secured with benzoin.

1. Dressing Compositions

a. Group 1 rhPDGF Dosage

As described in Regranex Package Insert, “each square centimeter ofulcer surface area will require approximately 0.25 cm length of gelsqueezed from 15 gram tube”. Formula: (1×w)÷4=cm length Regranex. For a1.5 cm×1.5 cm square wound: (1.5×1.5)÷4=0.56 cm length Regranex. Asdescribed in Regranex Package Insert, “the weight of Regranex gel from15 g tube is 0.25 g/cm length”. Regranex is 0.01% rhPDGF-BB or 100 μg/gRegranex. For 0.56 cm length of product, the weight of product is 0.14 gfor a total dose of PDGF-BB of 14 μg. For sites treated with Regranexfor 21 days the maximum total dose for the study period (assuming nochange in open wound size from Day 0) would be 14 μg/day×21 days or 294μg of PDGF-BB. However, at Days 7 and 14 the open wound size wasdetermined for all Regranex treated sites and the amount of Regranexapplied was recalculated using the formula above ([1×w]÷4=cm lengthRegranex).

b. Group3 rhPDGF and Group 2 Saline Dosages

The concentration of rhPDGF-BB used in the study was 0.3 mg/ml or 300μg/ml. To not exceed a total dose for the study period of 294 μg PDGF-BB(same total maximum study dose as Regranex), a total of 0.98 ml of 0.3mg/ml PDGF-BB would be applied to the wound site over the 21-day studyperiod. Assuming a total of 3 administrations (days 0, 7 and 14), eachadministration would consist of ≈327 μl PDGF-BB onto the collagen spongerepresenting a dose of approximately 98 μg PDGFBB/administration(slightly more than 7× the initial individual dose for Regranex treatedsites). This represents a total of 145 μl per square centimeter of openwound surface area (327 μl/2.25 cm² wound surface area).

The volume of 0.3 mg/ml PDGF-BB (Group 3) or buffer/sterile saline(Group 2) to be applied to the new collagen sponge on Days 7 and 14 wasdetermined using the following formula:

145×cm² open wound surface area (length [cm]×width [cm] of open wound).

c. Collagen Sponge

As described above for Group 1 treated sites, all wounds were evaluatedand measured at Days 7 and 14 to record the open wound measurements foreach individual site. For sites treated with a collagen sponge (Group 2and Group 3), the sponge was measured and trimmed to fit the open woundportion of the original wound following removal of the dressing, gentlerinsing of the site and documentation of findings including measurementsand photographic documentation.

C. Surgical Procedures

On Day 0, animals were anesthetized with isoflurane. The hair on theback was clipped and the skin swabbed with an aseptic solution. Atemplate was used to mark a 1.5×1.5 cm square on the mid-back of theanimal and a full thickness wound, corresponding to the template, wasmade by excising the skin and the panniculus carnosus. A hot watercirculation pad or equivalent was placed under the animal to maintainnormal body temperature during procedures, and animals recovered on asimilar hot water circulation pad. Buprenorphine (0.06 mg/kg) was givenby subcutaneous injections immediately after recovery from anesthesiaand every 12 hours thereafter for 72 hours. Warmed Ringers solution (0.5mL) was given by sub-cutaneous injection after the mice have recoveredconsciousness. The wounding of the animal was carried out under asepticconditions. The wound site was photographed and the length and widthmeasured immediately after excision and daily thereafter using a digitalcaliper. From Days 0 to 21, mice were administered test articles aslisted in Table 1.

D. Study Results

1. Animal Survival

Three animals died or were prematurely euthanized during this study (allanimals from Group 1—Regranex). The first animal was found dead one dayafter surgery (Animal #3). The second animal (Animal #1) had to besacrificed on Day 5 due to self-mutilating the rear flank posterior tothe wound site. Animal #5 in Group 1 had to be sacrificed on day 16 as aresult of losing more than 20% of its starting body weight. Thefollowing Table 2 summarizes the animal deaths/sacrifice:

TABLE 2 Summary of Animal Deaths/Sacrifice Day 1 Day 6 Day 16 Group 1,Animal #3 Group 1, Animal #1 Group 1, Animal #5 Found Dead Sacrified,Exceeded 20% Weight Loss Self Mutilating

2. Wound Measurements

The wound area was measured using a digital caliper and the length (L)and width (W) of each wound was recorded. Wound area was calculatedusing the formula to calculate the area of a square where A=L×W. FIG. 1shows the area of the wounds for each group on Days 0, 7, 14 and 21.Peak wound area was recorded on Day 0 for all three groups withsubsequent decreases in mean wound area on Day 7, Day 14, and Day 21.All treated groups showed a substantial decrease in wound area duringthe course of the study.

To provide an additional measurement and account for wounds that may notof healed in the shape of a square (and therefore not be captured in theformula used above), the inside of the wounds were also measured bytracing the inside wound edge using ImageJ Software™. FIG. 2 representsthe wound area from each animal using this approach for each treatmentgroup. FIGS. 3-6 show the average wound area for all treatment groups byevaluation day (Day 0, Day 7, Day 14, and Day 21) as a scatter plot toprovide a more detailed assessment of the individual measurementsrecorded on those days. For animals that died during the study, the lastdata point is carried forward in FIGS. 3-6. FIGS. 7A-7B highlights a keyaspect of the invention, namely that positive results are achieved withfewer applications of the therapeutic composition. FIG. 7A shows theamount of wound area reduction (mm²) per cumulative number of treatmentsat each of the four time points (Day 0, Day 7, Day 14, and Day 21) forGroups 1 and 3. FIGS. 8A-8D show the average percent of wound closureover the course of the study for each Group. For animals that died, thelast data point was carried forward.

3. Clinical Assessments

Wound images were also clinically assessed for possible differences inthe degree of healing with respect to reepithelialization and formationof granulation tissue. Representative images of the wounds from eachanimal at each time point are shown in FIGS. 9-12 from Day 0 (FIG. 9),Day 7 (FIG. 10), Day 14 (FIG. 11) and Day 21 (FIG. 12). The raw imagesfrom each treatment show that Group 3 (rhPDGF/collagen sponge group)resulted in a demonstrable acceleration in the formation of granulationtissue and re-epithelialization compared to Group 2 (the collagen spongecontrol group treated with buffer). In addition, wounds treated withRegranex daily (Group 1) also showed a better wound closure ratecompared to Group 2 (the buffer control+collagen). Histopathology onformalin fixed samples of the wound areas was also performed and furthercorroborated accelerated wound healing resulting from treatment with therhPDGF/collagen and Regranex treated wounds compared to buffer controltreated sponges. The pathology also suggests even further improvement inre-epithelialization in wounds treated with rhPDGF/collagen overRegranex treated.

Representative samples of the histopathological samples are provided inFIGS. 13-18 which include a series of photomicrographs of a crosssection of the wound site on day 21 from three study animals, includingGroup 1—Animal 2 (FIGS. 17 and 18), Group 2—Animal 2 (FIGS. 13 and 14),and Group 3—Animal 1 (FIGS. 15 and 16). For each set of photomicrographsthere is shown a 2× (FIGS. 13, 15, and 17) and a 10× magnification(FIGS. 14, 16, and 18).

Regarding Group 1, FIGS. 17 and 18 show that the wound was 100%resurfaced at Day 21, although some shearing was apparent due to thefragility of the dermal-epidermal architecture. Arrow 60 in FIG. 17indicates the approximate location of the adjacent epidermis to the leftand the wound bed to the right. FIG. 18 provides a higher magnificationimage from the middle of the wound. 100% epidermal resurfacing isapparent and the presence of differentiated stratification in theepidermis indicates its maturity. The neodermis still contains a highdensity of capillaries and new collagen formation is underway.

Regarding Group 2, the photomicrographs illustrate that a portion of thecollagen sponge persisted in the wound bed for 21 days. In this woundedge the sponge appeared to obstruct the resurfacing of the epithelium.The arrow 10 in FIG. 13 indicates the wound edge. As seen in FIG. 13,some granulation tissue developed beneath the collagen sponge. Referringto FIG. 14, the collagen sponge and the granulation tissue are indicatedby arrows 20 and 30, respectively. As shown, the collagen spongeappeared adherent in this region although infiltration with cells isminimal. A classic granulation tissue is shown formed beneath thesponge.

Regarding Group 3, FIGS. 15 and 16 illustrate the wound is 100%resurfaced with epidermis. The arrow 40 in FIG. 15 indicates theapproximate edge of the wound bed, and the region 50 is subcutaneousfat. FIG. 16 was taken within the middle of the wound, and shows thatall evidence of the original collagen sponge is gone. The wound is 100%resurfaced and is well stratified with a stratum corneum indicatingmaturity. The neodermis shows evidence of new collagen production andthe cellularity is decreased indicating that the dermal tissue ismaturing and losing the immature characteristics of granulation tissue.

E. Study Conclusions

The following conclusions were made from this study:

-   (1) 21 applications of Regranex were given in Group 1, while only 3    applications of buffer/collagen or rhPDGF/collagen wound dressings    were applied in Groups 2 and 3, respectively.-   (2) Three animals from Group 1 (Regranex) were either found dead or    had to be euthanized during the in-life portion of the study.-   (3) All treated groups showed a decrease in wound area from Day 0-21    as determined by both caliper measurements and wound tracing using    ImageJ software analysis. At sacrifice (Day 21), 2 of 5 Regranex    treated wounds, 3 of 5 rhPDGF/collagen treated wounds and 0 of 5    collagen dressing treated wounds were healed.-   (4) The raw images from each treatment show that Group 3    (rhPDGF/collagen) results in a demonstrable acceleration in the    formation of granulation tissue and re-epithelialization compared to    the collagen wound dressing control group treated with buffer (Group    2). In addition, wounds treated with Regranex daily (Group 1) also    showed a better closure rate compared to the control collagen sponge    treated animals.-   (5) Healing as assessed by wound reepithelialization was greatest in    wounds treated with three applications of rhPDGF/collagen (Group 3)    compared to 21 applications of Regranex (Group 1) or three    applications of the collagen wound dressing wetted with saline.-   (6) Three (3) weekly applications of rhPDGF/collagen (Group 3)    accelerate wound closure, including granulation tissue formation and    re-epithelialization compared to a collagen wound dressing (Group 2)    and appear at least as effective as 21 daily doses of Regranex Gel    (Group 1).-   (7) rhPDGF/collagen is safe and effective, promoting better healing    of diabetic wounds compared to the marketed collagen wound dressing.    3 of 5 rhPDGF/collagen treated wounds completely healed, as    evidenced by complete re-epithelialization, compared to 0 of 5    collagen wound dressing treated animals.-   (8) rhPDGF/collagen is safe and effective, promoting angiogenesis,    granulation tissue formation and re-epithelialization compared to a    marketed collagen wound dressing as demonstrated histologically.-   (9) rhPDGF/collagen, a sterile product, is highly biocompatible as    demonstrated histologically.-   (10) rhPDGF/collagen is much easier to apply than Regranex Gel,    which should improve patient compliance.-   (11) rhPDGF/collagen may be safer than Regranex, given that animals    that received Regranex had a high mortality rate, while no such    mortality was observed with rhPDGF/collagen or the collagen wound    dressing.

Example 2—Prophetic

A study is conducted to demonstrate the efficacy of the noveltherapeutic compositions and wound treatment methods described herein.The same study design outlined for Example 1 is also used for this studyincluding the db/db mouse model with five test groups—a standard of caregroup (saline moistened gauze), a Regranex group, a collagen spongegroup, and two groups utilizing treatment compositions in accordancewith the present invention comprising PDGF-BB and a collagen sponge. Asdetailed below, however, the frequency of the dosing is changed in thisstudy. The study is also designed so that the total dose of PDGFdelivered over the course of the study in both the Regranex group andthe collagen sponge/PDGF-BB groups is the same.

A. Experimental Design

The experimental design is refined by the results of the study describedin Example 1, however it is anticipated that the number of animals pergroup are greater (i.e. eight) and the study duration is longer, i.e. 28days. Additionally the test and control collagen+/−PDGF articles areadministered topically as wound dressings immediately following theinduction of the wound and at about Day 14 for a total of twoapplications (Group 5) or immediately after surgery and at Days 7, 14and 21 for a total of four applications of test articles in accordancewith the novel compositions and treatment methods described herein (seeTable X). Negative control (saline moistened gauze) and Regranex treatedsites will undergo 28 daily administrations topically as wound dressingin accordance with the prescribed Instructions for Use.

In this Example, blood glucose levels are determined prior to the startof the study and again just prior to sacrifice on Day 28 to confirmdiabetic disease state. In all other aspects, the design for this studyis the same as described in Example 1. The study details are outlined inbelow in Table 3.

TABLE 3 Example 2 Study Design Dressing Changes & Group Number of Route/Wound Number Animals Wound Treatment Frequency Assessment 1 8 male Day 0Saline Moistened Topical/28 Daily 27; Daily To (db/db) 1.5 cm × Gauzeapplications Day 28 1.5 cm 2 8 male Day 0 Regranex Gel Topical/28 Daily27; Daily To (db/db) 1.5 cm × applications Day 28 1.5 cm 3 8 male Day 0Collagen Sponge Topical/4 applications 3; Days 7, 14, (db/db) 1.5 cm ×Days 0, 7, 14 and 21 21 and 28* 1.5 cm 4 8 male Day 0 Collagen Sponge +Topical/4 applications 3; Days 7, 14, (db/db) 1.5 cm × 326 μl 0.3 mg/mlDays 0, 7, 14 and 21 21 and 28* 1.5 cm PDGF-BB 5 8 male Day 0 CollagenSponge + Topical/2 applications 1; Day 14, 21^(#) (db/db) 1.5 cm × 654μl 0.3 mg/ml Days 0 and 14 and 28* 1.5 cm PDGF-BB *Wound assessment andnecropsy; ^(#)Wound assessment only.

Every day for the period of the study, each animal is inspected and itssurvival recorded, in order to assess possible visual differences inanimal responses among treatment groups. The rate of wound closure willbe determined, as will the percentage of wounds completely healed at anygiven time point.

B. PDGF-BB Dose Calculations

The PDGF dosage used in this study is designed to mimic the actualtherapeutic doses in accordance with either the present invention(Groups 4 and 5) or the actual therapeutic dose prescribed in accordancewith the Regranex label (Group 2). The dosage for Group 2 is determinedin the same manner as the Regranex group in Example 1. For sites treatedwith Regranex for 28 days the total dose of PDGF administered for thestudy period is 14 μg/day×28 days or 392 μg of PDGF-BB. This dose mayalso be expressed amount of PDGF per area of the original wound size(“area dose”) of 6.22 μg/cm2/day or 174.2 μg/cm2 of PDGF-BB.

For Groups 4 and 5, a PDGF solution is used having a PDGF concentrationof 0.3 mg/ml or 300 μg/ml. To achieve a total dose for the study periodof 392 μg PDGF-BB (the same total study dose as in the Regranex group),a total of 1.307 ml PDGF solution is applied to the wound site over the28 day study period. For sites receiving dressing changes once every 7days there are a total of 4 administrations (days 0, 7, 14 and 21). Eachadministration consists of 327 μl PDGF-BB (or buffer alone) onto thecollagen sponge. With respect to dose of PDGF, each administrationconsists of 0.3 μg/μl×326 μl or 98 ug PDGF-BB (approximately 7× Regranexindividual dose). For sites receiving just two doses, at Day 0 and 14there are a total of 2 administrations. Each administration consists of654 μl PDGF-BB onto the collagen sponge. Each administration wouldconsist of 0.3 μg/μl×654 μl or 196 ug PDGF-BB (approximately 14×Regranex individual dose). These doses may also be expressed as amountof PDGF per area of the original wound size (“area dose”). With respectto Group 4, the PDGF dose is 43.5 μg/cm2/dose or 174 μg/cm2 of PDGF-BB.With respect to Group 5, the PDGF dose is about 87 μg/cm2/dose or 174μg/cm2 of PDGF, i.e. the same cumulative dose in all groups but Groups 4and 5 have many fewer doses.

Example 3—Prophetic

A randomized clinical trial is conducted to assess the effectiveness ofvarious compositions of rhPDGF-BB and collagen as compared to standardof care (consisting of moist wound healing with removal of excess woundexudate, debriding necrotic tissue, off-loading of pressure, salinemoistened gauze, antibiotics if needed and wound dressing) and Regranexin the treatment of chronic diabetic foot ulcers. Table 4 belowsummarizes the study design. For each arm of the study (1-37) theproduct is applied at the dosage and frequency indicated in Table 4 forup to 20 weeks or until complete wound closure. Regranex is applied inaccordance with its approve US labeling. rhPDGF-BB/collagen compositionsare applied in accordance with the procedures (steps 1-5) describedabove in paragraph 50.

The outcome measures for the study are:

-   -   Incidence of complete wound closure.    -   Time to achieve complete wound closure.    -   Percentage reduction in total ulcer surface area at each visit.    -   Number of ulcer recurrence observed 12 weeks after wound        healing.    -   Treatment emergent adverse events (up to 52 weeks).

The inclusion criteria for the study include:

-   -   Men or women aged 18 years old or older, with type 1 or 2        diabetes mellitus    -   Patient with a single ulcer on the treated feet    -   Patient able and willing to provide informed consent    -   Patient able and willing to comply with protocol visits and        procedure    -   Patient willing to use an off-loading method during the whole        duration of the study    -   Full-thickness plantar, lateral or dorsal ulcer of the extremity        (below the malleolus), excluding inter-digits ulcer (web        spaces), extending through the epidermis and dermis, but not        involving bone, tendons, ligaments or muscles (grade IA as        defined by University of Texas Diabetic Wound Classification or        Grade 1 according to Wagner classification)    -   Chronic ulcer of at least six weeks despite appropriate wound        care    -   Ulcer area measured with the formula Length×Width×0.8 following        sharp debridement, of 1 to 10 cm², both inclusive    -   Well controlled infection or cellulitis (systemic        antibiotherapy) before Baseline Visit    -   Peripheral neuropathy as assessed by Semmes-Weinstein        monofilament test or by the bio esthesimeter (vibration        perception threshold)    -   Ankle brachial pressure index >0.60 and <1.3    -   Women surgically sterile, post-menopausal, or agree to practice        adequate contraception and have a negative pregnancy test at        screening    -   Non-nursing

The exclusion criteria for the study include:

-   -   Inter digit ulcers    -   Ulcer of other cause or origin: electrical, chemical or        radiation insult, bedsores, vascular ulcer or Charcot        deformities ulcers    -   Charcot foot    -   Wound originated from amputation bed    -   Active ulcer infection assessed by clinical examination and        radiography if necessary. Presence of necrosis, purulence or        sinus tracts that cannot be removed by debridement and        controlled by standard wound care    -   Active osteomyelitis affecting the area of the target ulcer    -   Poorly controlled diabetes (uncontrolled glycemia: HbAlc        %>=10%), renal failure (serum creatinine >3.0 mg/dL), poor        nutritional status (albumin <3.0 g/dL or total protein <6.5        g/dL)    -   Known connective tissue or malignant disease    -   Concomitant treatment with corticosteroids, immunosuppressive        agents, radiation therapy, or anticancer chemotherapy    -   Use of investigational drug/device or growth factor within 30        days    -   Topical application of any advance wound care on this wound        (antiseptics, antibiotics, debriders, enzyme) within 7 days    -   Vascular reconstruction within 8 weeks    -   Patients expected to be noncompliant with the protocol (not        available for the duration of the trial, treatment or wound care        compliance), or felt to be unsuitable by the Investigator for        any other reason    -   A history of severe cerebrovascular events

TABLE 4 Example 4 Study Design Max Initial Later ratio rhPDGF TreatmentNumber of dose doses Dosage solution/Collagen Frequency Treatments(μg/cm2) (μg/cm2) Adjust (μl/cm3) 1 Regranex daily 140 6.25 6.25 weekly2 Standard of Care daily 140 — — — 3 rhPDGF/collagen 3 days 46 10 10 6days 67 4 sponge 3 days 46 18.75 18.75 6 days 125 5 5 days 28 31.2531.25 10 days 208 6 7 days 20 43.75 43.75 7 days 292 7 14 days 10 87.587.5 14 days 583 8 21 days 6 131.25 131.25 21 days 875 9 28 days 5 175175 28 days 1167 10 35 days 4 218.75 218.75 35 days 1459 11 42 days 3262.5 262.5 42 days 1750 12 3 days 46 10 10 6 days 134 13 3 days 4618.75 18.75 6 days 250 14 5 days 28 31.25 31.25 10 days 416 15 7 days 2043.75 43.75 7 days 584 16 14 days 10 87.5 87.5 14 days 1166 17 21 days 6131.25 131.25 21 days 1750 18 28 days 5 175 175 28 days 2334 19 35 days4 218.75 218.75 35 days 2918 20 42 days 3 262.5 262.5 42 days 3500 21 3days 46 20 10 6 days 67 22 3 days 46 37.5 18.75 6 days 125 23 5 days 2862.5 31.25 10 days 208 24 7 days 20 87.5 43.75 7 days 292 25 14 days 10175 87.5 14 days 583 26 21 days 6 262.5 131.25 21 days 875 27 28 days 5350 175 28 days 1167 28 35 days 4 437.5 218.75 35 days 1459 29 42 days 3525 262.5 42 days 1750 30 3 days 46 18.75 18.75 6 days 250 31 5 days 2831.25 31.25 10 days 370 32 7 days 20 43.75 43.75 7 days 480 33 14 days10 87.5 87.5 14 days 890 34 21 days 6 131.25 131.25 21 days 1290 35 28days 5 175 175 28 days 1690 36 35 days 4 218.75 218.75 35 days 2100 3742 days 3 262.5 262.5 42 days 2500

Each of the rhPDGF/collagen sponge compositions performs better thanRegranex or standard of care in at least one of the outcome measures,and/or achieves a substantially equivalent result with the applicationof less cumulative rhPDGF applied over the treatment period or theapplication of fewer treatments which leads to better patientcompliance.

The embodiments, variations, and sequences described herein shouldprovide an indication of the utility and versatility of the presentinvention. Other embodiments that do not provide all of the features andadvantages set forth herein may also be utilized, without departing fromthe spirit and scope of the present invention. Such modifications andvariations are considered to be within the scope of the invention.

1. A method of treating a wound that extends through the epidermis,wherein said method comprises: (1) debriding the wound to removenecrotic or infected tissue; (2) forming a therapeutic compositionconsisting essentially of sterile recombinant human PDGF-BB (rhPDGF-BB)in a physiologic solution and a sterile porous biocompatible carrier,wherein the porous biocompatible carrier is a collagen sponge orcollagen wound dressing, and said therapeutic composition is free froman enzyme inhibitor; (3) applying the therapeutic composition to thewound surface in an amount that is at least about 10 μg rh PDGF-BB percm² of treated wound surface area, wherein the carrier provides asubstrate for cell attachment and vascular ingrowth as the wound heals;(4) covering the wound with a dressing; and (5) monitoring the healingof the wound during a treatment period and repeating steps (1)-(4) toretreat the wound at treatment intervals of 7 or more days, (6) whereinthe wound is retreated from 2 to 20 times, and wherein each retreatmentcomprises applying the therapeutic composition to the wound surface inan amount that is at least 10 μg rhPDGF-BB/cm² treated wound surfacearea up to 100 μg rhPDGF-BB/cm² treated wound surface area.
 2. Themethod of claim 1 wherein the wound extends through the epidermis for atleast six weeks duration.
 3. The method of claim 1 wherein the wound isan ulcer.
 4. The method of claim 1 wherein the wound is a diabetic footulcer.
 5. The method of claim 1 wherein the wound is a venous stasisulcer.
 6. The method of claim 1 wherein the wound is a pressure ulcer.7. The method of claim 1 wherein the step of forming the therapeuticcomposition comprises combining the sterile rhPDGF-BB and the sterileporous biocompatible carrier, wherein: (i) the sterile rhPDGF-BBcomprises an rhPDGF-BB solution comprising between about 0.05 mg/ml toabout 5 mg/ml of rhPDGF-BB; and (ii) the ratio of the rhPDGF-BB solutionto the carrier is between about 0.1 ml/cm3 carrier to about 1 ml/cm3 orthe ratio of rhPDGF-BB to the carrier is between about 75 μg PDGF/cm3 ofcarrier to about 225 μg PDGF/cm3 of carrier.
 8. The method of claim 1wherein the step of forming the therapeutic composition comprisescombining the sterile rhPDGF-BB and the sterile porous biocompatiblecarrier, wherein: (i) the sterile rhPDGF-BB comprises an rhPDGF-BBsolution comprising between about 0.05 mg/ml to about 5 mg/ml ofrhPDGF-BB, and (ii) the ratio of the rhPDGF-BB solution to the carrieris between about 0.25 ml solution/cm³ of carrier to about 5 mlsolution/cm³ of carrier, or the ratio of rhPDGF-BB to the carrier isbetween about 75 μg rhPDGF-BB/cm³ of carrier to about 750 μgrhPDGF-BB/cm³ of carrier.
 9. The method of claim 1 wherein at leastabout 80% of the rhPDGF-BB on a weight basis is unclipped rhPDGF-BB. 10.The method of claim 1 wherein the porous biocompatible carrier is acollagen sponge.
 11. The method of claim 1 wherein the porousbiocompatible carrier is a collagen wound dressing.
 12. The method ofclaim 1 wherein the cumulative total amount of rhPDGF-BB applied to thewound during the treatment period is less than about 50 mg of rhPDGF-BB.13. The method of claim 1 wherein the cumulative total amount ofrhPDGF-BB applied to the wound during the treatment period is less thanabout 25 mg of rhPDGF-BB.
 14. The method of claim 1 wherein the wound isretreated from 2 to 20 times over a maximum treatment period of 2 to 20weeks.
 15. The method of claim 1 wherein the steps (1)-(4) are repeatedto retreat the wound at a retreatment frequency of at least every 8days.
 16. The method of claim 1 wherein the steps (1)-(4) are repeatedto retreat the wound at a retreatment frequency of at least every 10days.
 17. The method of claim 1 wherein the wound is retreated a maximumof 10 times.
 18. The method of claim 1 wherein: (A) the wound extendsthrough the epidermis for at least six weeks duration; (B) the step offorming the therapeutic composition comprises combining the sterilerhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) thesterile rhPDGF-BB comprises an rhPDGF-BB solution comprising betweenabout 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, wherein at least about80% of the rhPDGF-BB on a weight basis is unclipped rhPDGF-BB, and (ii)the ratio of the rhPDGF-BB solution to the carrier is between about 0.1ml solution/cm³ of carrier to about 1 ml solution/cm³ of carrier, or theratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm³of carrier to about 225 μg rhPDGF-BB/cm³ of carrier; (C) the wound isretreated from 2 to 20 times over a maximum treatment period of 2 to 20weeks at a retreatment frequency of at least every 8 days; and (D) thecumulative total amount of rhPDGF-BB applied to the wound during thetreatment period is less than about 50 mg of rhPDGF-BB.
 19. A method oftreating a wound that extends through the epidermis, wherein said methodcomprises: (1) debriding the wound to remove necrotic or infectedtissue; (2) forming a therapeutic composition consisting essentially ofsterile recombinant human PDGF-BB (rhPDGF-BB) in a physiologic solutionand a sterile porous biocompatible carrier, wherein the porousbiocompatible carrier is a collagen sponge or collagen wound dressing;(3) applying the therapeutic composition to the wound surface in anamount that is at least about 10 μg rhPDGF-BB per cm² of treated woundsurface area, wherein the carrier provides a substrate for cellattachment and vascular ingrowth as the wound heals; (4) covering thewound with a dressing; and (5) monitoring the healing of the woundduring a treatment period and repeating steps (1)-(4) to retreat thewound at treatment intervals of 8 or more days, wherein the wound isretreated from 2 to 20 times over a maximum treatment period of 2 to 20weeks, and wherein each retreatment comprises applying the therapeuticcomposition to the wound surface in an amount that is at least 10 μgrhPDGF-BB/cm² treated wound surface area up to 100 μg rhPDGF-BB/cm²treated wound surface area.
 20. The method of claim 19 wherein the woundis a wound that extends through the dermis for at least six weeksduration.
 21. The method of claim 19 wherein the wound is an ulcer. 22.The method of claim 19 wherein the wound is a diabetic foot ulcer. 23.The method of claim 19 wherein the wound is a venous stasis ulcer. 24.The method of claim 19 wherein the wound is a pressure ulcer.
 25. Themethod of claim 19 wherein the step of forming the therapeuticcomposition comprises combining the sterile rhPDGF-BB and the sterileporous biocompatible carrier, wherein: (i) the sterile rhPDGF-BBcomprises an rhPDGF-BB solution comprising between about 0.05 mg/ml toabout 5 mg/ml of rhPDGF-BB, and (ii) the ratio of the rhPDGF-BB solutionto the carrier is between about 0.1 ml solution/cm³ of carrier to about1 ml solution/cm³ of carrier, or the ratio of rhPDGF-BB to the carrieris between about 75 μg rhPDGF-BB/cm³ of carrier to about 225 μgrhPDGF-BB/cm³ of carrier.
 26. The method of claim 19 wherein the step offorming the therapeutic composition comprises combining the sterilerhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) thesterile rhPDGF-BB comprises an rhPDGF-BB solution comprising betweenabout 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, and (ii) the ratio ofthe rhPDGF-BB solution to the carrier is between about 0.25 mlsolution/cm³ of carrier to about 5 ml solution/cm³ of carrier, or theratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm³of carrier to about 750 μg rhPDGF-BB/cm³ of carrier.
 27. The method ofclaim 19 wherein the cumulative total amount of rhPDGF-BB applied to thewound during the treatment period is less than about 50 mg of rhPDGF-BB.28. The method of claim 19 wherein the cumulative total amount ofrhPDGF-BB applied to the wound during the treatment period is less thanabout 25 mg of rhPDGF-BB.
 29. The method of claim 19 wherein the steps(1)-(4) are repeated to retreat the wound at a retreatment frequency ofat least every 10 days.
 30. The method of claim 19 wherein the wound isretreated a maximum of 10 times.
 31. The method of claim 19 wherein: (A)the wound extends through the epidermis for at least six weeks duration;(B) the step of forming the therapeutic composition comprises combiningthe sterile rhPDGF-BB and the sterile porous biocompatible carrier,wherein: (i) the sterile rhPDGF-BB comprises an rhPDGF-BB solutioncomprising between about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB,wherein at least about 80% of the rhPDGF-BB on a weight basis isunclipped rhPDGF-BB, and (ii) the ratio of the rhPDGF-BB solution to thecarrier is between about 0.1 ml solution/cm³ of carrier to about 1 mlsolution/cm³ of carrier, or the ratio of rhPDGF-BB to the carrier isbetween about 75 μg rhPDGF-BB/cm³ of carrier to about 225 μgrhPDGF-BB/cm³ of carrier; and (C) the cumulative total amount ofrhPDGF-BB applied to the wound during the treatment period is less thanabout 50 mg of rhPDGF-BB.
 32. A method of treating a wound that extendsthrough the epidermis: (1) debriding the wound to remove necrotic orinfected tissue; (2) forming a therapeutic composition consistingessentially of sterile recombinant human PDGF-BB (rhPDGF-BB) in aphysiologic solution and a sterile porous biocompatible carrier, whereinthe porous biocompatible carrier is a collagen sponge or collagen wounddressing; (3) applying the therapeutic composition to the wound surfacein an amount that is at least about 10 μg rhPDGF-BB per cm² of treatedwound surface area, wherein the carrier provides a substrate for cellattachment and vascular ingrowth as the wound heals; (4) covering thewound with a dressing; and (5) monitoring the healing of the woundduring a treatment period and repeating steps (1)-(4) to retreat thewound at treatment intervals of 10 or more days, wherein the wound isretreated from 2 to 20 times, and wherein each retreatment comprisesapplying the therapeutic composition to the wound surface in an amountthat is at least 10 μg rhPDGF-BB/cm² treated wound surface area up to100 μg rhPDGF-BB/cm² treated wound surface area.
 33. The method of claim32 wherein the wound extends through the epidermis for at least sixweeks duration.
 34. The method of claim 32 wherein the wound is anulcer.
 35. The method of claim 32 wherein the wound is a diabetic footulcer.
 36. The method of claim 32 wherein the wound is a venous stasisulcer.
 37. The method of claim 32 wherein the wound is a pressure ulcer.38. The method of claim 32 wherein the step of forming the therapeuticcomposition comprises combining the sterile rhPDGF-BB and the sterileporous biocompatible carrier, wherein: (1) the sterile rhPDGF-BBcomprises an rhPDGF-BB solution comprising between about 0.05 mg/ml toabout 5 mg/ml of rhPDGF-BB, and\ (2) the ratio of the rhPDGF-BB solutionto the carrier is between about 0.1 ml solution/cm³ of carrier to about1 ml solution/cm³ of carrier, or the ratio of rhPDGF-BB to the carrieris between about 75 μg rhPDGF-BB/cm³ of carrier to about 225 μgrhPDGF-BB/cm³ of carrier.
 39. The method of claim 32 wherein the step offorming the therapeutic composition comprises combining the sterilerhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) thesterile rhPDGF-BB comprises an rhPDGF-BB solution comprising betweenabout 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, and (ii) the ratio ofthe rhPDGF-BB solution to the carrier is between about 0.25 mlsolution/cm³ of carrier to about 5 ml solution/cm³ of carrier, or theratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm³of carrier to about 750 rhPDGF-BB/cm³ of carrier.
 40. The method ofclaim 32 wherein the cumulative total amount of rhPDGF-BB applied to thewound during the treatment period is less than about 50 mg of rhPDGF-BB.41. The method of claim 32 wherein the cumulative total amount ofrhPDGF-BB applied to the wound during the treatment period is less thanabout 25 mg of rhPDGF-BB.
 42. The method of claim 32 wherein the woundis retreated from 2 to 20 times over a maximum treatment period of 2 to20 weeks.
 43. The method of claim 1 wherein when the rhPDGF-BB and theporous carrier are combined, the carrier is capable of entrapping therhPDGF-BB within its pores such that the rhPDGF-BB is released over timeas the carrier is absorbed by the patient's body, thereby providingcontrolled delivery of rhPDGF-BB at the wound over an extended period oftime and simultaneously providing a matrix for new cell and tissueingrowth.
 44. A method of treating a wound that is a burn, wherein saidmethod comprises: (1) debriding the wound to remove necrotic or infectedtissue; (2) forming a therapeutic composition consisting essentially ofsterile recombinant human PDGF-BB (rhPDGF-BB) in a physiologic solutionand a sterile porous biocompatible carrier, wherein the porousbiocompatible carrier is a collagen sponge or collagen wound dressing,and said therapeutic composition is free from an enzyme inhibitor; (3)applying the therapeutic composition to the wound surface in an amountthat is at least about 10 μg rh PDGF-BB per cm² of treated wound surfacearea, wherein the carrier provides a substrate for cell attachment andvascular ingrowth as the wound heals; (4) covering the wound with adressing; and (5) monitoring the healing of the wound during a treatmentperiod and repeating steps (1)-(4) to retreat the wound at treatmentintervals of 7 or more days, (6) wherein the wound is retreated from 2to 20 times, and wherein each retreatment comprises applying thetherapeutic composition to the wound surface in an amount that is atleast 10 μg rhPDGF-BB/cm² treated wound surface area up to 100 μgrhPDGF-BB/cm² treated wound surface area.
 45. The method of claim 44wherein the step of forming the therapeutic composition comprisescombining the sterile rhPDGF-BB and the sterile porous biocompatiblecarrier, wherein: (i) the sterile rhPDGF-BB comprises an rhPDGF-BBsolution comprising between about 0.05 mg/ml to about 5 mg/ml ofrhPDGF-BB; and (ii) the ratio of the rhPDGF-BB solution to the carrieris between about 0.1 ml/cm3 carrier to about 1 ml/cm3 or the ratio ofrhPDGF-BB to the carrier is between about 75 μg PDGF/cm3 of carrier toabout 225 μg PDGF/cm3 of carrier.
 46. The method of claim 44 wherein thestep of forming the therapeutic composition comprises combining thesterile rhPDGF-BB and the sterile porous biocompatible carrier, wherein:(i) the sterile rhPDGF-BB comprises an rhPDGF-BB solution comprisingbetween about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, and (ii) theratio of the rhPDGF-BB solution to the carrier is between about 0.25 mlsolution/cm³ of carrier to about 5 ml solution/cm³ of carrier, or theratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm³of carrier to about 750 μg rhPDGF-BB/cm³ of carrier.
 47. The method ofclaim 44 wherein at least about 80% of the rhPDGF-BB on a weight basisis unclipped rhPDGF-BB.
 48. The method of claim 44 wherein the porousbiocompatible carrier is a collagen sponge.
 49. The method of claim 44wherein the porous biocompatible carrier is a collagen wound dressing.50. The method of claim 44 wherein the cumulative total amount ofrhPDGF-BB applied to the wound during the treatment period is less thanabout 50 mg of rhPDGF-BB.
 51. The method of claim 44 wherein thecumulative total amount of rhPDGF-BB applied to the wound during thetreatment period is less than about 25 mg of rhPDGF-BB.
 52. The methodof claim 44 wherein the wound is retreated from 2 to 20 times over amaximum treatment period of 2 to 20 weeks.
 53. The method of claim 44wherein the steps (1)-(4) are repeated to retreat the wound at aretreatment frequency of at least every 8 days.
 54. The method of claim44 wherein the steps (1)-(4) are repeated to retreat the wound at aretreatment frequency of at least every 10 days.
 55. The method of claim44 wherein the wound is retreated a maximum of 10 times.
 56. The methodof claim 44 wherein: (A) the wound is a burn of a least six weeksduration; (B) the step of forming the therapeutic composition comprisescombining the sterile rhPDGF-BB and the sterile porous biocompatiblecarrier, wherein: (i) the sterile rhPDGF-BB comprises an rhPDGF-BBsolution comprising between about 0.05 mg/ml to about 5 mg/ml ofrhPDGF-BB, wherein at least about 80% of the rhPDGF-BB on a weight basisis unclipped rhPDGF-BB, and (ii) the ratio of the rhPDGF-BB solution tothe carrier is between about 0.1 ml solution/cm³ of carrier to about 1ml solution/cm³ of carrier, or the ratio of rhPDGF-BB to the carrier isbetween about 75 μg rhPDGF-BB/cm³ of carrier to about 225 μgrhPDGF-BB/cm³ of carrier; (C) the wound is retreated from 2 to 20 timesover a maximum treatment period of 2 to 20 weeks at a retreatmentfrequency of at least every 8 days; and (D) the cumulative total amountof rhPDGF-BB applied to the wound during the treatment period is lessthan about 50 mg of rhPDGF-BB.
 57. A method of treating a wound that isa burn, wherein said method comprises: (1) debriding the wound to removenecrotic or infected tissue; (2) forming a therapeutic compositionconsisting essentially of sterile recombinant human PDGF-BB (rhPDGF-BB)in a physiologic solution and a sterile porous biocompatible carrier,wherein the porous biocompatible carrier is a collagen sponge orcollagen wound dressing; (3) applying the therapeutic composition to thewound surface in an amount that is at least about 10 μg rhPDGF-BB percm² of treated wound surface area, wherein the carrier provides asubstrate for cell attachment and vascular ingrowth as the wound heals;(4) covering the wound with a dressing; and (5) monitoring the healingof the wound during a treatment period and repeating steps (1)-(4) toretreat the wound at treatment intervals of 8 or more days, wherein thewound is retreated from 2 to 20 times over a maximum treatment period of2 to 20 weeks, and wherein each retreatment comprises applying thetherapeutic composition to the wound surface in an amount that is atleast 10 μg rhPDGF-BB/cm² treated wound surface area up to 100 μgrhPDGF-BB/cm² treated wound surface area.
 58. The method of claim 57wherein the step of forming the therapeutic composition comprisescombining the sterile rhPDGF-BB and the sterile porous biocompatiblecarrier, wherein: (i) the sterile rhPDGF-BB comprises an rhPDGF-BBsolution comprising between about 0.05 mg/ml to about 5 mg/ml ofrhPDGF-BB, and (ii) the ratio of the rhPDGF-BB solution to the carrieris between about 0.1 ml solution/cm³ of carrier to about 1 mlsolution/cm³ of carrier, or the ratio of rhPDGF-BB to the carrier isbetween about 75 μg rhPDGF-BB/cm³ of carrier to about 225 μgrhPDGF-BB/cm³ of carrier.
 59. The method of claim 57 wherein the step offorming the therapeutic composition comprises combining the sterilerhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) thesterile rhPDGF-BB comprises an rhPDGF-BB solution comprising betweenabout 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, and (ii) the ratio ofthe rhPDGF-BB solution to the carrier is between about 0.25 mlsolution/cm³ of carrier to about 5 ml solution/cm³ of carrier, or theratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm³of carrier to about 750 μg rhPDGF-BB/cm³ of carrier.
 60. The method ofclaim 57 wherein the cumulative total amount of rhPDGF-BB applied to thewound during the treatment period is less than about 50 mg of rhPDGF-BB.61. The method of claim 57 wherein the cumulative total amount ofrhPDGF-BB applied to the wound during the treatment period is less thanabout 25 mg of rhPDGF-BB.
 62. The method of claim 57 wherein the steps(1)-(4) are repeated to retreat the wound at a retreatment frequency ofat least every 10 days.
 63. The method of claim 57 wherein the wound isretreated a maximum of 10 times.
 64. The method of claim 57 wherein: (A)the wound is a burn of at least six weeks duration; (B) the step offorming the therapeutic composition comprises combining the sterilerhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) thesterile rhPDGF-BB comprises an rhPDGF-BB solution comprising betweenabout 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, wherein at least about80% of the rhPDGF-BB on a weight basis is unclipped rhPDGF-BB, and (ii)the ratio of the rhPDGF-BB solution to the carrier is between about 0.1ml solution/cm³ of carrier to about 1 ml solution/cm³ of carrier, or theratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm³of carrier to about 225 μg rhPDGF-BB/cm³ of carrier; and (C) thecumulative total amount of rhPDGF-BB applied to the wound during thetreatment period is less than about 50 mg of rhPDGF-BB.
 65. A method oftreating a wound that is a burn: (1) debriding the wound to removenecrotic or infected tissue; (2) forming a therapeutic compositionconsisting essentially of sterile recombinant human PDGF-BB (rhPDGF-BB)in a physiologic solution and a sterile porous biocompatible carrier,wherein the porous biocompatible carrier is a collagen sponge orcollagen wound dressing; (3) applying the therapeutic composition to thewound surface in an amount that is at least about 10 μg rhPDGF-BB percm² of treated wound surface area, wherein the carrier provides asubstrate for cell attachment and vascular ingrowth as the wound heals;(4) covering the wound with a dressing; and (5) monitoring the healingof the wound during a treatment period and repeating steps (1)-(4) toretreat the wound at treatment intervals of 10 or more days, wherein thewound is retreated from 2 to 20 times, and wherein each retreatmentcomprises applying the therapeutic composition to the wound surface inan amount that is at least 10 μg rhPDGF-BB/cm² treated wound surfacearea up to 100 μg rhPDGF-BB/cm² treated wound surface area.
 66. Themethod of claim 65 wherein the step of forming the therapeuticcomposition comprises combining the sterile rhPDGF-BB and the sterileporous biocompatible carrier, wherein: (1) the sterile rhPDGF-BBcomprises an rhPDGF-BB solution comprising between about 0.05 mg/ml toabout 5 mg/ml of rhPDGF-BB, and\ (2) the ratio of the rhPDGF-BB solutionto the carrier is between about 0.1 ml solution/cm³ of carrier to about1 ml solution/cm³ of carrier, or the ratio of rhPDGF-BB to the carrieris between about 75 μg rhPDGF-BB/cm³ of carrier to about 225 μgrhPDGF-BB/cm³ of carrier.
 67. The method of claim 65 wherein the step offorming the therapeutic composition comprises combining the sterilerhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) thesterile rhPDGF-BB comprises an rhPDGF-BB solution comprising betweenabout 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, and (ii) the ratio ofthe rhPDGF-BB solution to the carrier is between about 0.25 mlsolution/cm³ of carrier to about 5 ml solution/cm³ of carrier, or theratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm³of carrier to about 750 μg rhPDGF-BB/cm³ of carrier.
 68. The method ofclaim 65 wherein the cumulative total amount of rhPDGF-BB applied to thewound during the treatment period is less than about 50 mg of rhPDGF-BB.69. The method of claim 65 wherein the cumulative total amount ofrhPDGF-BB applied to the wound during the treatment period is less thanabout 25 mg of rhPDGF-BB.
 70. The method of claim 65 wherein the woundis retreated from 2 to 20 times over a maximum treatment period of 2 to20 weeks.
 71. The method of claim 44 wherein when the rhPDGF-BB and theporous carrier are combined, the carrier is capable of entrapping therhPDGF-BB within its pores such that the rhPDGF-BB is released over timeas the carrier is absorbed by the patient's body, thereby providingcontrolled delivery of rhPDGF-BB at the wound over an extended period oftime and simultaneously providing a matrix for new cell and tissueingrowth.